Anticoagulants, Antiplatelets, and Thrombolytics (Methods in Molecular Biology)

ME T H O D S IN MO L E C U L A R BI O L O G Y Series Editor John M. Walker School of Life Sciences University of Her...

Author: Shaker A. Mousa

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ME T H O D S

IN

MO L E C U L A R BI O L O G Y

Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For other titles published in this series, go to www.springer.com/series/7651

TM

Anticoagulants, Antiplatelets, and Thrombolytics Second Edition

Edited by

Shaker A. Mousa Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Rensselaer, NY, USA

Editor Shaker A. Mousa Albany College of Pharmacy and Health Sciences Pharmaceutical Research Institute One Discovery Drive Rensselaer, NY 12144 USA [email protected]

ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-60761-802-7 e-ISBN 978-1-60761-803-4 DOI 10.1007/978-1-60761-803-4 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2010929846 © Springer Science+Business Media, LLC 2003, 2010 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)

Preface The past 2 decades have witnessed significant advances in the discovery and development of novel drugs to prevent and treat thromboembolic disorders, such as oral direct antiXa and anti-IIa (thrombin) antagonists, as well as oral antiplatelet ADP antagonists with rapid onset and offset. The introduction of direct oral factor Xa and thrombin inhibitors that do not require monitoring and have no significant food or drug interactions represents a significant advance that may lead to the replacement of oral warfarin and injectable heparin or low molecular weight heparin (LMWH) in some, but probably not all, indications. In addition, there has been concentrated effort aimed at identifying novel uses of traditional antithrombotic drugs as well as combinations of agents, such as more than one antiplatelet, or antiplatelet plus anticoagulant. These tremendous achievements have resulted in improved management of arterial and venous thromboembolic-associated disorders. Although the morbidity and mortality resulting from acute coronary disease has been reduced by more than 50% over the past 30 years, it is reasonable to anticipate further reductions of similar magnitude in the decade ahead. Advances in our understanding of the mechanisms of pathogenesis of venous thromboembolism (VTE), acute coronary syndromes, cerebral vascular ischemia, and diseases associated with thrombotic events have provided critical insight for the development of various therapeutic approaches to control these pathogenic events. The roles of plasmatic proteins, blood cells, vascular endothelium, and target organs in thrombogenesis are becoming more clear. Identification of endogenous inhibitors of thrombogenesis such as antithrombin III, tissue factor pathway inhibitor (TFPI), protein C, prostacyclin, nitric oxide, and physiologic activators of fibrinolysis has led to the development of both direct and indirect modalities to treat thrombosis. Knowledge of the proteases involved in thrombogenesis, as well as tissue factor, coagulation factors, adhesion molecules, and fibrinolytic inhibitors, has provided additional insight into the mechanisms by which thrombogenesis can be pharmacologically controlled. All of these novel strategies could not have happened without the utilization of key in vitro and in vivo clinically relevant experimental models for the screening and evaluation of these novel antiplatelets, anticoagulants, and thrombolytics (discussed in Chapters 1 and 2). Newly developed anti-Xa agents are characterized by high affinity and selectivity for Xa as compared to other serine proteases. In addition to their inhibitory effects on plasmatic coagulation processes, including thrombin generation, thrombin-mediated platelet reactions, and clot-bound pro-thrombinase complexes, there is evidence that some of these agents might interfere with receptor-mediated intracellular signaling events induced by factor Xa that regulate proliferation of vascular smooth muscle cells and other cells. The current outlook for anti-Xa agents is that they have the potential to become important prophylactic and treatment drugs for various venous thromboembolic disorders as well as adjuvants to other antithrombotic therapies in arterial thrombosis. Major advances in the development of oral anticoagulants are progressing very well, with the goal of developing safe and effective oral anticoagulants that do not require frequent monitoring or dose adjustment and that have minimal food/drug interactions.

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Vitamin K antagonist, with its inherent limitations of multiple food and drug interactions and frequent need for monitoring, remains the only oral anticoagulant currently approved for long-term secondary thromboprophylaxis in VTE. The oral direct thrombin inhibitor ximelagatran was withdrawn from the world market due to safety concerns. Newer anticoagulant drugs such as injectable pentasaccharides (e.g., idraparinux, SSR126517E), oral direct thrombin inhibitors (e.g., dabigatran), oral direct factor Xa inhibitors (e.g., rivaroxaban, apixaban, YM-150, DU-176b), and tissue factor/factor VIIa complex inhibitors are “tailor-made” to target specific pro-coagulant complexes and have the potential to greatly expand oral antithrombotic targets for both acute and long-term treatment of VTE, acute coronary syndromes, and prevention of stroke in atrial fibrillation patients (discussed in Chapter 5). The oral direct factor Xa inhibitor rivaroxaban represents a potentially attractive alternative to warfarin as it may enable simplified once daily dosing, appears to require no therapeutic monitoring, and has lower potential for drug interactions. At present, the safety and efficacy of rivaroxaban for the prophylaxis and treatment of VTE have been evaluated in phase-II and phase-III trials involving over 24,000 patients. In addition, rivaroxaban is currently being evaluated for the treatment of pulmonary embolism, secondary prevention after acute coronary syndromes, and the prevention of stroke and non-central nervous system embolism in patients with non-valvular atrial fibrillation. Several other oral direct anti-Xa inhibitors are in advanced clinical development, approved in Europe and under FDA review for the prevention and treatment of thromboembolic disorders (discussed in Chapter 6). Dabigatran is a novel oral direct reversible (fast onset and offset) thrombin inhibitor that binds to both free and clot-bound thrombin with a high affinity and specificity. Dabigatran has predictable and reproducible pharmacokinetics that are not affected by interactions with food. It is not metabolized by CYP450, does not induce nor inhibit CYP450, resulting in low potential for drug interactions, and does not require coagulation or platelet monitoring. The RE-NOVATE trial demonstrated that oral dabigatran etexilate at fixed doses is a well-tolerated alternative to injectable enoxaparin for the prevention of VTE after total knee replacement. The RELY trial demonstrated that oral dabigatran etexilate concurrently reduces both thrombotic and hemorrhagic events at two different doses (150 and 110 mg BID), exhibiting different and complimentary advantages over warfarin. At a dose of 150 mg BID, dabigatran had superior efficacy with similar bleeding, while at a dose of 110 mg BID, there was significantly less bleeding with similar efficacy in patients with atrial fibrillation at risk of stroke. Based on the accumulating clinical evidence, dabigatran represents the future of anticoagulation in the prevention and treatment of venous and arterial thrombosis alone and in conjunction with current antiplatelets and thrombolytics. Anti-platelet therapies remain a major focus in drug development. While aspirin is still considered the gold standard for antiplatelet therapy because of its high benefit-to-cost and benefit-to-risk ratios, ADP receptor antagonists, including ticlopidine, clopidogrel, and prasugrel, represent significant additions to aspirin in the management of different forms of arterial thromboembolic disorders (Chapter 7). Prasugrel is a novel thienopyridine that inhibits the platelet P2Y12 receptor and provides more rapid and consistent platelet inhibition than clopidogrel (Chapter 8). It is becoming clear, however, that there is variability in individual responses to antiplatelet agents such as clopidogrel, which may limit their widespread implementation. Various definitions of “non-responders” to antiplatelet therapy (i.e., aspirin resis-

Preface

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tance) tend to compound this issue. Aspirin resistance refers to aspirin-treated patients that are insensitive to aspirin treatment based on ex vivo tests of platelet activation and who experience recurrent cardiovascular disease. Estimates of aspirin resistance based on these criteria range from 20 to 80%, indicating that ex vivo tests are not an optimal tool for such assessments. In long-term aspirin-treated patients, there is evidence of low level but functionally relevant platelet thromboxane A2 formation, which was responsible for enhanced platelet activation in response to platelet agonists. These studies, however, did not fully exclude aspirin compliance, which could be a factor in such a phenomenon. Two trials performed in patients with coronary artery disease demonstrated that laboratory evidence of aspirin resistance was not detectable when aspirin compliance was accurately monitored. The same phenomenon was reported for other anti-platelet drugs such as clopidogrel. Given the multi-factorial nature of atherothrombosis, recurrence of cardiovascular events in aspirin-treated patients is not necessarily suggestive of drug failure. A cause-effect relationship between platelet insensitivity to aspirin and cardiovascular recurrence has not been defined overall because aspirin compliance has rarely been considered. Until such crucial information is taken into account, it would be prudent to take into consideration the distinction between “clinical resistance” to aspirin and resistance to taking the drug. To carefully define anti-platelet resistance, issues such as dose levels, standardized monitoring parameters, drug-drug interactions, and drug monitoring to document compliance should be addressed in future studies. The next decade should see considerable attention focused on the vascular endothelium, which occupies a strategic position at the interface between tissue and blood. The normal endothelium releases multiple antiplatelet, anti-inflammatory, thrombolytic and vasodilator molecules, such as prostacyclin and nitric oxide, which are potent inhibitors of platelet and monocyte activation and function as vasodilators. In addition, the normal endothelial surface expresses other protective molecules, including ecto-ADP, which degrades ADP, leading to inhibition of platelet aggregation; thrombomodulin, which activates protein C; and heparin-like molecules, which serve as cofactors for antithrombin III and heparin. The normal endothelium also secretes tissue plasminogen activator, which activates fibrinolysis. Insult or injury to the endothelium is accompanied by loss of these protective molecules and induction of expression of adhesive, pro-coagulant and pro-inflammatory molecules, vasoconstrictors, and mitogenic factors, leading to the development of thrombosis, smooth muscle cell migration and proliferation, and atherosclerosis. Hence, protective mechanisms of endothelial function represent new frontiers in the prevention and treatment of thromboembolic disorders that will have minimal effect on hemostasis. Improved understanding of the cell biology of plaque instability and endothelial hemostasis will promote a number of novel therapeutic strategies, including passivation of the endothelium, reduction of low-density lipoprotein (LDL) in the vessel wall (through decreasing serum LDL levels or accelerating reverse cholesterol transport), inhibition of LDL oxidation, thereby raising high density lipoprotein (HDL), and inhibition of inflammatory cytokine expression, as well as inhibition of thrombus formation upstream in the coagulation cascade or inhibition of activation of coagulation. The recognition that thrombotic disorders represent a syndrome rather than a disease is of crucial importance in the development of newer drugs. Either a poly-therapeutic approach with drug combinations or a drug with multiple actions will likely be more appropriate for the management of thrombotic disorders.

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Preface

This second edition of Anticoagulants, Antiplatelets, and Thrombolytics provides updates on various strategies in thrombosis, experimental models, and clinical and recent advances in the discovery and development of novel antithrombotics. Future directions in the coming decade should focus on the prevention of thromboembolic disorders and the protection of the vascular endothelium. Shaker A. Mousa

Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1.

In Vitro Methods of Evaluating Antithrombotics and Thrombolytics . . . . . . . Shaker A. Mousa

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2.

In Vivo Models for the Evaluation of Antithrombotics and Thrombolytics . . . . Shaker A. Mousa

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3.

Heparin and Low-Molecular Weight Heparins in Thrombosis and Beyond . . . . 109 Shaker A. Mousa

4.

Laboratory Methods and Management of Patients with Heparin-Induced Thrombocytopenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 Margaret Prechel, Walter P. Jeske, and Jeanine M. Walenga

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Novel Anticoagulant Therapy: Principle and Practice . . . . . . . . . . . . . . . 157 Shaker A. Mousa

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Oral Direct Factor Xa Inhibitors, with Special Emphasis on Rivaroxaban . . . . . 181 Shaker A. Mousa

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Antiplatelet Therapies: Drug Interactions in the Management of Vascular Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 Shaker A. Mousa

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Prasugrel: A Novel Platelet ADP P2Y12 Receptor Antagonist . . . . . . . . . . . 221 Shaker A. Mousa, Walter P. Jeske, and Jawed Fareed

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Antithrombotic Effects of Naturally Derived Products on Coagulation and Platelet Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 Shaker A. Mousa

10.

Assessment of Anti-Metastatic Effects of Anticoagulant and Antiplatelet Agents Using Animal Models of Experimental Lung Metastasis . . . . . . . . . 241 Ali Amirkhosravi, Shaker A. Mousa, Mildred Amaya, Todd Meyer, Monica Davila, Theresa Robson, and John L. Francis

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Adhesion Molecules: Potential Therapeutic and Diagnostic Implications . . . . . 261 Shaker A. Mousa

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Pharmacogenomics in Thrombosis . . . . . . . . . . . . . . . . . . . . . . . . 277 Shaker A. Mousa

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Diagnosis and Management of Sickle Cell Disorders . . . . . . . . . . . . . . . 291 Shaker A. Mousa and Mohamad H. Qari

Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309

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Contributors MILDRED AMAYA • Florida Hospital Center for Thrombosis Research, Orlando, FL, USA ALI AMIRKHOSRAVI • Florida Hospital Center for Thrombosis Research, Orlando, FL, USA MAMDOUH BAKHOS • Department of Thoracic and Cardiovascular Surgery, Stritch School of Medicine, Loyola University, Maywood, IL, USA MONICA DAVILA • Florida Hospital Center for Thrombosis Research, Orlando, FL, USA JAWED FAREED • Department of Pathology, Stritch School of Medicine, Loyola University, Maywood, IL, USA JOHN L. FRANCIS • Florida Hospital Center for Thrombosis Research, Orlando, FL, USA WALTER P. JESKE • Department of Thoracic & Cardiovascular Surgery, Stritch School of Medicine, Loyola University, Maywood, IL, USA TODD MEYER • Florida Hospital Center for Thrombosis Research, Orlando, FL, USA SHAKER A. MOUSA • Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Rensselaer, NY, USA MARGARET PRECHEL • Department of Pathology, Stritch School of Medicine, Loyola University, Maywood, IL, USA MOHAMAD H. QARI • College of Medicine, King Abdul-Aziz University, Jeddah, Saudi Arabia THERESA ROBSON • Florida Hospital Center for Thrombosis Research, Orlando, FL, USA JEANINE M. WALENGA • Department of Thoracic & Cardiovascular Surgery, Stritch School of Medicine, Loyola University, Maywood, IL, USA

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Chapter 1 In Vitro Methods of Evaluating Antithrombotics and Thrombolytics Shaker A. Mousa Abstract Platelets play a crucial role in primary hemostasis by forming hemostatic plugs at sites of vascular injury. There is abundant evidence that platelets also play a pivotal role in the pathogenesis of arterial thrombotic disorders, including unstable angina (UA), myocardial infarction (MI), and stroke. The underlying pathophysiological mechanism of these processes has been recognized as the disruption or erosion of a vulnerable atherosclerotic plaque, leading to local platelet adhesion and subsequent formation of partially or completely occlusive platelet thrombi. A variety of methods have been used to assess platelet aggregation, blood coagulation, and the ex vivo and/or in vitro efficacy of platelet antagonists, anticoagulants, and thrombolytics. Key words: Platelets, aggregation, coagulation, thrombosis, in vitro models, adhesion.

1. Introduction The specific platelet surface receptors that support the initial adhesive interactions that ultimately lead to the formation of thrombi are determined by the local fluid dynamics of the vasculature and the extracellular matrix constituents exposed at the sites of vascular injury. Under high shear conditions, the adhesion of un-activated platelets to exposed sub-endothelial surfaces of atherosclerotic or injured vessels is mediated by binding of platelet glycoprotein (GP) Ib/IX/V complex to collagen and von Willebrand factor (vWF) presented on exposed vessel surfaces (1, 2). This primary adhesion to the matrix activates platelets, ultimately resulting in platelet aggregation, which is mediated predominantly by the binding of adhesive proteins, such as fibrinogen S.A. Mousa (ed.), Anticoagulants, Antiplatelets, and Thrombolytics, Methods in Molecular Biology 663, DOI 10.1007/978-1-60761-803-4_1, © Springer Science+Business Media, LLC 2003, 2010

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and vWF, to GPIIb/IIIa. Direct platelet aggregation in the bulk phase under conditions of abnormally elevated fluid shear stress, analogous to what occurs in atherosclerotic or constricted arterial vessels, may also be important (3). Shear-induced platelet aggregation is dependent upon the availability of vWF and the presence of GPIb/IX and GPIIb/IIIa on the platelet membrane. It has been postulated that under high shear stress conditions, the interaction of vWF with the GPIb/IX complex is the initial event leading to platelet activation and also triggers the binding of vWF to GPIIb/IIIa to induce platelet aggregate formation. The coagulation cascade consists of a complex network of interactions resulting in thrombin-mediated conversion of fibrinogen to fibrin, a major component of the thrombus. Initiation of the coagulation cascade occurs through the release of thromboplastin (tissue factor) and subsequent activation and conversion of factor VII into tissue factor/factor VIIa complex (“exogenous pathway”), or by the so-called contact activation pathway or “endogenous pathway,” which proceeds through factors XII, XI, and IX to the assembly of a tenase complex, consisting of activated factors VIII and IX and Ca2+ , on phospholipid membranes. Both the exogenous and endogenous tenase complex can activate factor X, which induces the formation of the prothrombinase complex, consisting of factor Xa, factor Va, and Ca2+ , on phospholipid surfaces. Assembly of the prothrombinase complex leads to the activation of thrombin, which cleaves fibrinogen to yield fibrin.

2. In Vitro Coagulation Tests 2.1. Blood Coagulation Tests 2.1.1. Purpose and Rational

Three coagulation tests, prothrombin time (PT), activated partial thromboplastin time (aPTT), and thrombin time (TT), can differentiate between exogenous and endogenous pathway effects and distinguish them from effects on fibrin formation. Typically, the influence of compounds on plasmatic blood coagulation is determined by measuring PT, aPTT, and TT ex vivo.

2.1.2. Procedure

Male Sprague-Dawley rats weighing 200–220 g are administered test compound, or vehicle as a control, through an oral, intraperitoneal, or intravenous route. After a period of time for absorption (adsorption time), animals are anesthetized by intravenous injection of sodium pentobarbital (60 mg/kg). The caudal caval vein is exposed by midline incision and 1.8 ml of blood is collected into

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a plastic syringe containing 0.2 ml of 100 mM citrate buffer, pH 4.5 (Behring Werke, Marburg, DE). The sample is immediately agitated and then subjected to centrifugation in a plastic tube at 1,500×g for 10 min, after which plasma is collected and transferred to a clean plastic tube. Coagulation tests for TT, PT, and aPTT should be performed within 3 hours (h). In general, citrated plasma coagulates upon the addition of the appropriate compound (see below), and time to clot formation (coagulation time) is determined using a coagulometer (Schnittger and Gross, Amelung, Brake, DE). For the detailed laboratory diagnosis of bleeding disorders and assessment of blood coagulation, see Palmer (4) and Nilsson (5). PT. PT measures effects on the exogenous pathway of coagulation. Citrated plasma (0.1 ml) is incubated for 1 min at 37◦ C, R at which point 0.2 ml of human thromboplastin (Thromborel ; Behringwerke) is added. The coagulometer is started, and time to clot formation is recorded. aPTT. aPTT measures effects on the endogenous pathway of coagulation. Citrated plasma (0.1 ml) is mixed with 0.1 ml of R human placenta lipid extract (Pathrombin ; Behringwerke), and ◦ the mixture is incubated for 2 min at 37 C. Coagulation is initiated by the addition of 0.1 ml of 25 mM calcium chloride, at which point the coagulometer is started and time to clot formation is recorded. TT. TT measures effects on fibrin formation. Citrated plasma (0.1 ml) is mixed with 0.1 ml of diethyl barbiturate–citrate buffer, pH 7.6 (Behringwerke), and the mixture is incubated for 1 min at 37◦ C. Bovine test thrombin (0.1 ml) (30 IU/ml; Behringwerke) is added, at which point the coagulometer is started, and the time to clot formation is recorded. 2.2. Thrombelastography 2.2.1. Purpose and Rational

Thrombelastography (TEG) was developed by Hartert in 1948 (6). The thrombelastograph is a device that provides a continuous recording of the process of blood coagulation and subsequent clot retraction. Blood samples are transferred to cuvettes and maintained at 37◦ C. The cuvettes are set in motion around their vertical axes. Initially, a mirror suspended by a torsion wire in the plasma remains immobile as long as the plasma is fluid. There is a dynamic interplay between the cuvette and the mirror as fibrin forms, resulting in the transmission of motion within the cuvette to the mirror. The mirror will oscillate, the amplitude of which is governed by the specific mechanical properties of the clot, and reflect light onto a thermo-paper recording. Modern thrombelastographs translate the light recording into a digital signal that can be readily analyzed using a computer program.

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2.2.2. Procedure

TEG can be performed with whole blood or citrated plateletrich or platelet-poor plasma after re-calcification. Blood samples are obtained from test animals (i.e. Beagle dogs, 12–20 kg; rabbits, 1.7–2.5 kg; Wistar rats, 150–300 g; or humans). Test subjects receive the compound of interest by intravenous (iv), subcutaneous (sc) or oral administration. Ten- or twenty-min post dosing (iv or sc administration), or 60-, 90- or 180-min post dosing (oral administration), blood is collected. The blood samples are mixed with 3.8% trisodium citrate solution (1:9 citrate solution:blood) as an anticoagulant. Citrated whole blood is recalcified by the addition of 0.4 ml of isotonic calcium chloride. An aliquot (0.36 ml) of re-calcified whole blood is transferred to a pre-warmed cup of the thrombelastograph. After the apparatus has been correctly adjusted and the samples have been sealed with liquid paraffin to prevent drying, the start time for the procedure is noted, and the thrombelastogram is recorded for 2 h.

2.2.3. Evaluation

The following measurements are the standard variables of TEG: Reaction time (r). The time from sample placement in the cup until onset of clotting (defined as an amplitude of 1 mm). This represents the rate of initial fibrin formation. Clot formation time (k). The difference from the 1 mm r to 20 mm amplitude. k represents the time for a fixed degree of viscoelasticity to be achieved by the clot formation due to fibrin buildup and cross linking. Alpha angle (α ◦ ). Angle formed by the slope of the TEG tracing from the r to k value. It denotes the speed at which solid clot forms. Maximum amplitude (MA). Greatest amplitude on the TEG trace. MA represents the absolute strength of the fibrin clot and is a direct function of the maximum dynamic strength of fibrin and platelets. Clot strength (G; in dynes per square centimeter). It is defined by G = (5,000MA)/(96–MA). In tissue factor-modified TEG (7), clot strength is clearly a function of platelet concentration. Lysis 30, Lysis 60 (Ly30, Ly60). Reduction of amplitude relative to MA at 30 and 60 min after the time of MA. These parameters represent the influence of clot retraction and fibrinolysis. Readers are referred to a number of studies in which TEG has been instrumental in advancing the field of antithrombotics and thrombolytics. Most recently, TEG has been used to analyze the effects of a variety of stimuli on platelet/fibrin clot dynamics (8). In other work, Bhargava et al. (9) used TEG to compare the anticoagulant effects of a new potent heparin preparation and then commercially available heparin in vitro using citrated dog and human blood. Barabas et al. (10) used the fibrin plate

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assay and TEG to assess the anti-fibrinolytic effects of synthetic thrombin inhibitors. Scherer et al. (11) described a short-time, endotoxin-induced rabbit model of hyper-coagulability for the study of the coagulation cascade, enabling the analysis of coagulation inhibitors and their therapeutic effects by a number of techniques, including TEG. In 1997, Khurana et al. (7) introduced tissue factor-modified TEG to study platelet GPIIb/IIIa function, establishing a quantitative assay of platelet function. Using this modification to TEG, Mousa et al. (8) identified two classes of GPIIb/IIIa antagonists, one with high binding affinity for resting and activated platelets and a slow platelet dissociation rate (class I) that exhibited potent inhibition of platelet function, and one with a fast platelet dissociation rate (class II). TEG was also used in a phase-II clinical trial to assess the efficacy of an oral platelet GPIIb/IIIa antagonist on platelet/fibrin clot dynamics (12). Zuckerman et al. (13) compared TEG with other common coagulation tests (fibrinogen, PT, aPTT, platelet count, and fibrin split products) and found a strong correlation between thrombelastographic variables and common laboratory tests. Moreover, TEG had a higher sensitivity for blood clotting anomalies. TEG also provides additional information on the hemostatic process. In contrast to most laboratory assays, in which the end point is the formation of the first fibrin strands, TEG measures the coagulation process from the initiation of clotting to the final stages of clot lysis and retraction. Another advantage of TEG is that it allows the use of whole non-anticoagulated blood without the influence of citrate or other anticoagulants. 2.3. Chandler Loop 2.3.1. Purpose and Rational

The Chandler loop technique measures the generation of in vitro thrombi in a moving column of blood (14). Thrombi generated in the Chandler device are morphologically similar to human thrombi formed in vivo (15), with platelet-rich upstream sections (“white heads”) that are relatively resistant to tissue plasminogen activator(t-PA)-mediated thrombolysis as compared to red blood cell-rich downstream components (“red tails”) (16).

2.3.2. Procedure

One millimeter of non-anticoagulated whole blood is drawn directly into a polyvinyl tube 25 cm in length with an internal diameter of 0.375 cm (1 mm=9.9-cm tubing). The two ends of the tube are then brought together and closed using an outside plastic collar. The circular tube is placed at the center of a turntable, tilted to an angle of 23º, and then rotated at 17 rpm. When the developing thrombus inside the tube becomes large enough to occlude the lumen, the blood column becomes static and begins to move on the table in the direction of rotation.

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Stringer et al. (16) used this method to determine the influence of an anti-plasminogen activator inhibitor (PAI)-1 antibody (CLB-2C8) on t-PA-induced lysis of Chandler thrombi in vitro. Investigators used citrated blood supplemented with 5.8 μM [125 I]-labeled fibrinogen prior to re-calcification. Thrombi generated in the Chandler loop were washed with isotonic saline and then cut transversally into an upstream (head) and a downstream (tail) part, and then each part was analyzed using a gamma counter to determine pre-values. The head and tail were then subjected to thrombolysis by the addition of 300 μl of phosphatebuffered saline (PBS) containing plasminogen (2 μM) and t-PA (0.9 nM). Over a period of 240 min, aliquots (10 μl) from each section were removed at 30, 60, 120, 180, and 240 min, and radioactivity was determined. Radioactivity at each time point as compared to the pre-value was then expressed as a percentage of clot lysis. In 1998, Van Giezen et al. (17) used this method to successfully differentiate the effects of an anti-PAI-1 polyclonal antibody (PRAP-1) on human and rat thrombi. 2.4. Platelet Aggregation and De-aggregation in Platelet-Rich Plasma or Washed Platelets (Born Method) 2.4.1. Purpose and Rational

Contact between non-activated platelets and exposed subendothelial tissue leads to adhesion through two main mechanisms: (1) at high shear rates, binding of sub-endothelial vWF to platelet GPIb–IX–V complex and (2) binding of collagen to integrin α2β1 and GPVI. Platelet adhesion initiates several processes, including shape changes, secretion, and the activation of GPIIb/IIIa ligand binding sites, resulting in the formation of platelet aggregates. Activation of GPIIb/IIIa is also achieved through receptor cross-signaling initiated by the binding of a number of agonists to G-protein-coupled receptors. To measure platelet aggregation, one of the following agonists is added to platelet-rich plasma (PRP) or washed platelets (WP): ADP, arachidonic acid (which is converted to thromboxane A2) or the thromboxane agonist U46619, collagen, thrombin or thrombin receptor-activating peptide (TRAP), serotonin, epinephrine, or platelet activating factor (PAF). Upon stirring, the formation of platelet aggregates is monitored photometrically as changes in optical density, typically for 4 min. This test was developed by Born (18, 19) and is used to quantitatively evaluate the effect of compounds on the induction of platelet aggregation in vitro or ex vivo. For in vitro studies, human PRP is the preferred starting material.

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2.4.2. Procedure

For ex vivo assays, mice, rats, or guinea pigs of either sex receive test compound, or vehicle as a control, by oral, intraperitoneal (ip), or iv administration. At the end of absorption time, blood is collected by caval venipuncture under pentobarbital sodium anesthesia with xylazine (8 mg/kg intramuscular) premedication. From rabbits (Chinchilla, 3 kg in weight), blood is drawn by cardiopuncture under xylazine (20 mg/kg intramuscular) sedation. A control blood sample is collected before administration of the test compound, and the second sample is drawn at the end of absorption time. For in vitro assays of human blood, samples are collected from the vein of adult volunteers who have not received any medication during the 2 weeks prior to collection.

2.4.2.1. Preparation of PRP, Platelet-poor Plasma (PPP), and WP

The entire procedure is performed in plastic (polystyrene) tubes and carried out at room temperature. Freshly collected venous blood is anticoagulated with hirudin (1:9, hirudin:blood) or anticoagulant citrate dextrose (ACD) solution (1:9, ACD:blood) and then subjected to centrifugation at 170×g for 15 min to obtain PRP. The PRP supernatant is carefully removed, and the sample is subjected to centrifugation again at 1,500×g for 10 min to obtain PPP. PRP is diluted with PPP to a platelet count of 3×108 /ml before use in the aggregation assay. To obtain WP, 8.5 volumes of human blood are collected into 1.5 volumes of ACD and then subjected to centrifugation, as described for PRP. PRP is acidified to a pH of 6.5 by the addition of ACD (approximately 1 ml per 10 ml of PRP). Acidified PRP is subjected to centrifugation for 20 min at 430×g, and then the pellet is re-suspended to the original volume in Tyrode’s solution (120 mM NaCl, 2.6 mM KCl, 12 mM NaHCO3 , 0.39 mM NaH2 PO4 , 10 mM HEPES, 5.5 mM Glucose, and 0.35% albumin) and diluted to a platelet count of 3×108 /ml. The assay should be completed within 3 h of blood collection. For ex vivo assays, duplicate samples of PRP (320 μl) from drug-treated and vehicle control subjects (for rabbits, the control sample is taken before drug administration) are inserted into the aggregometer at 37◦ C under continuous magnetic stirring at 1,000 rpm. After the addition of 40 μl of physiological saline and 40 μl of aggregating agent, changes in optical density are monitored continuously at 697 nm. For in vitro assays, 40 μl of the test solution is added to 320 μl of PRP or WP from untreated subjects. The samples are inserted into the aggregometer and incubated at 37◦ C for 2 min under continuous magnetic stirring at 1,000 rpm. After the addition of 40 μl of aggregating agent, changes in optical density are monitored continuously at 697 nm for 4 min, or until aggregation values are constant. In cases of thrombin activation of PRP, glycine–proline–aspartate–proline (GPRP) peptide is added

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in order to prevent fibrin formation. To measure de-aggregation, experimental compounds are added to stimulated PRP at 70% or 100% of control aggregation, and samples are monitored for 10 min. De-aggregation is measured as the decrease in light transmission over time (20). 2.4.3. Evaluation 2.4.3.1. For In Vitro Assays

1. Percent inhibition of platelet aggregation is determined in each concentration group relative to the respective vehicle control. 2. IC50 values are determined from non-linear fitting of the concentration–effect relationship curve. IC50 is defined as the concentration of test drug that achieves half-maximal inhibition of aggregation. 3. Percent de-aggregation is determined 10 min after the addition of compound; IC50 is calculated from the concentration–effect curve 4. Statistical significance is evaluated by means of the unpaired Student’s t-test.

2.4.3.2. For Ex Vivo Assays

1. The mean values for aggregation in each dosage group are compared to the respective vehicle control group (for rabbits, the control is before drug administration). 2. ED50 values are determined from the dose–response curves. ED50 is defined as the dose of drug that achieves halfmaximal inhibition of aggregation in animals. 3. Statistical significance is evaluated by means of the Student’s t-test (paired for rabbits; unpaired for others).

2.4.4. Critical Assessment of the Method

This assay, introduced by Born in 1926, has become a standard method in the clinical diagnosis of platelet function disorders and aspirin intake. Furthermore, the method is widely used in the discovery of antiplatelet drugs. The advantages of the method include the ability to rapidly measure a functional parameter in intact human platelets. However, processing of platelets during the preparation of PRP, WP, or filtered platelets from whole blood can result in platelet activation and separation of large platelets.

2.4.5. Modifications of the Method

Several authors have described modifications of the Born assay. Breddin et al. (21) described the use of a rotating cuvette to measure spontaneous aggregation of platelets from vascular patients. Klose et al. (22) measured platelet aggregation under laminar flow conditions using a thermo-regulated cone-plate streaming chamber in which shear rates were continuously augmented and platelet aggregation was measured based on light transmission through a transillumination system. Marguerie et al. (23, 24)

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developed a method of measuring two phases of platelet aggregation after gel filtration of a platelet suspension (see below). Lumley and Humphrey (25) described a method to measure platelet aggregation in whole blood (see below). Fratantoni and Poindexter (26) used a microtiter plate reader with specific modifications of sample agitation to measure platelet aggregation. A comparison of the 96-well microtiter plate method and conventional aggregometry revealed similar dose–response curves for thrombin, ADP, and arachidonic acid. Ammit and O’Neil (27) used a quantitative bioassay of platelet aggregation for rapid and selective measurement of PAF. 2.5. Platelet Aggregation After Gel Filtration 2.5.1. Purpose and Rational

Triggering of platelet activation by low concentrations of ADP, epinephrine, or serotonin, the so-called weak platelet agonists, in plasma- and fibrinogen-free platelet suspensions does not result in platelet aggregation unless exogenous fibrinogen is added. In contrast, platelet aggregation induced by thrombin, collagen, or prostaglandin endoperoxide, so-called strong agonists, is independent of exogenous fibrinogen because these substances induce the secretion of intracellular platelet ADP and fibrinogen. Analysis of platelet aggregation in gel-filtered platelet samples is carried out in cases when fibrinogen or vWF is needed in a defined concentration, or when plasma proteins could negatively interfere with the effects of compounds. The assay is used primarily to evaluate the influence of compounds on platelet integrin GPIIb/IIIa or other integrins or on platelet GPIb–IX–V.

2.5.2. Procedure 2.5.2.1. Preparation of Gel-Filtered Platelets

The entire procedure is performed in plastic (polystyrene) tubes at room temperature (24). Blood is drawn from healthy adult volunteers who have received no medication in the 2 weeks prior to collection. Venous blood (8.4 ml) is collected into 1.4 ml of ACD solution and subjected to centrifugation for 10 min at 120×g. PRP is carefully removed, the pH is adjusted to 6.5 with ACD solution, and the sample is subjected to centrifugation again at 285×g for 20 min. The resulting pellet is re-suspended in Tyrode’s buffer (approx. 500 μl of buffer/10 ml of PRP), and the platelet suspension is applied immediately to a Sepharose CL 2B column. Equilibration and elution (flow rate, 2 ml/min) are done with Tyrode’s buffer without hirudin and apyrase. Platelets are recovered in the void volume. The platelet suspension is adjusted to a final cell concentration of 4×108 /ml. Gel-filtered platelets (GFP) are kept at room temperature for 1 h before the assay is started.

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2.5.2.2. Experimental Course

For aggregation studies, GFP in Tyrode’s buffer are incubated with CaCl2 (final concentration, 0.5 mM) with or without fibrinogen (final concentration, 1 mg/ml) in polystyrene tubes. After 1 min, 20 μl of the test compound, or vehicle as a control, are added and the sample is incubated for an additional 2 min. Platelet agonist is added (20 μl), and changes in light transmission are recorded. The entire procedure is done under continuous magnetic stirring at 37◦ C (1,000 rpm) in the aggregometer. Samples with CaCl2 but without fibrinogen confirm that plasma proteins have been properly filtered out if neither spontaneous aggregation nor aggregation in the presence of weak agonists occurs; full aggregation of GFP in response to 10 μM ADP confirms that platelets are intact (i.e., minor pre-activation by gel filtration). Readers are referred to several references for a detailed methodology and evaluation of different agents by gel filtration platelet aggregation (23, 24, 28).

2.6. Platelet Aggregation in Whole Blood 2.6.1. Purpose and Rational

This method uses a whole blood platelet counter, which counts single platelets and does not require separation of platelets from other blood cell types. Platelet aggregation is induced in anticoagulated human whole blood samples by addition of the aggregating agents arachidonic acid or collagen. The number of platelets is determined in drug-treated and vehicle control samples and the percent inhibition of aggregation and IC50 values are calculated for each dosage group. This test system enables assessment of the effect of compounds on other blood cells, which can indirectly influence platelet aggregation. The method was originally described by Lumley and Humphrey (25) and Cardinal and Flower (29).

2.6.2. Procedure

The entire procedure is performed in plastic (polystyrene) tubes and is carried out at room temperature. Blood is drawn from healthy adult volunteers who have not received medication during the 2 weeks prior to collection. Venous blood (9 ml) is anticoagulated with 1 ml of sodium citrate and maintained in a closed tube at room temperature for 30–60 min until the start of the test. For aggregation analysis, 10 μl of compound, or vehicle as a control, are added to 480 μl of citrated blood. Samples in closed tubes are pre-incubated for 5 min in a 37◦ C water shaker bath with shaking (75 strokes/min). Aggregating agent (10 μl) is added and samples are incubated for another 10 min. The number of platelets (platelet count) is determined in aliquots of 10 μl immediately before and 10 min after the addition of aggregating

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agent (initial platelet count and 10-min platelet count, respectively) using a hemocytometer. The following samples are prepared in duplicate: 1. Control aggregation/spontaneous aggregation: 480 ml blood + 20 ml vehicle. Samples with >20% spontaneous aggregation should not be used to test for induced aggregation. 2. Maximum aggregation: 480 ml blood + 10 ml vehicle + 10 ml aggregating agent. Represents the maximum induced aggregation rate. 3. Test substance aggregation: 480 ml blood + 10 ml test substance + 10 ml aggregating agent. 2.7. Platelet Microand Macro-Aggregation Using Laser 2.7.1. Purpose and Rational

A new platelet aggregometer (AG-10; Kowa, Japan) that uses a laser light-scattering beam was introduced in 1996 by Tohgi et al. (30). This technique is a highly sensitive method of studying platelet aggregation based on the measurement of mean radius or particle size, making it possible to record the kinetics of formation of micro- and macro-aggregates in real time. Sensitivity in measurement of spontaneous aggregation is higher than in routine light transmittance. Platelet aggregate size, derived from the total voltage of lightscatter intensity at 1.0-second (s) intervals over a 10-min period, can be divided into three ranges: small (diameter 9–25 μm), medium (26–50 μm), and large (>50 μm) aggregates. Using laser scatter aggregation, it was found that young smokers had an increased number of small platelet aggregates, which were undetectable using a conventional aggregometer and the turbidometric method (31). The light-scatter aggregometer can detect platelet aggregation in the small-aggregate size range after the addition of unfractionated heparin (UFH), and the aggregates are dissociated upon incubation with protamine sulfate. Analysis of platelet aggregation induced by 0.5 U/ml of UFH in 36 normal subjects with no history of heparin exposure revealed 13 subjects with a positive response in excess of 0.5 V of light intensity in the small-aggregate size range. In chronic hemodialysis patients who had used heparin regularly for many years, a positive response, that is, the detection of heparin-induced aggregates, was observed in 37 of 59 patients, an increase over normal subjects. Light intensity in the small-aggregate size range was also enhanced during heparinized dialysis. In patients with a positive heparin response, the intensity of aggregate formation after heparin was significantly higher than heparin non-responders. Using the same system, it

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has been shown that enhanced platelet aggregation response to heparin is not inhibited by aspirin or argatroban, but can be inhibited by anti-GPIIb/IIIa antibodies. Enhanced platelet aggregation during heparin infusion was observed without the addition of ADP or TRAP using laser aggregometry (32). Limitations. This technique cannot be applied to whole blood, but can be used with PRP, WP, or GFP. 2.8. Fibrinogen Receptor Binding 2.8.1. Purpose and Rational

This assay is used to evaluate the binding characteristics of drugs that target the fibrinogen receptor. A single concentration of radioligand (125 I-fibrinogen, 30–50 nM) is incubated with human GFP in the presence of increasing concentrations of a non-labeled test drug (0.1 nM–1 mM). If the test drug binds to the fibrinogen receptor, it will compete with the radiolabeled ligand for receptor binding sites. Generally, the higher the affinity of the test drug for the receptor, the lower the concentration required to compete for binding, and the more potent the test drug will be. Platelets are activated by 10 mM ADP to stimulate 125 I-fibrinogen binding to the GPIIb/IIIa receptor.

2.8.2. Procedure 2.8.2.1. Preparation of GFP

Blood is collected from a healthy volunteer (200 ml). An aliquot (8.4 ml) is mixed with 1.4 ml of ACD buffer in a polystyrol tube and then subjected to centrifugation at 1,000 rpm for 15 min. The resulting PRP is collected and an aliquot is removed for a platelet count. Ten ml of PRP are mixed with 1 ml of ACD buffer to yield ACD–PRP, pH ∼6.5, and then 5 ml portions of ACD– PRP are transferred to plastic tubes and subjected to centrifugation at 1,600 rpm for 20 min. The resulting supernatants are decanted, and each pellet is re-suspended in 500 μl of Tyrode buffer C. An aliquot is removed for a platelet count to determine platelet loss, and then the platelet suspension is transferred to a Sepharose column that has been pre-equilibrated with approximately 100 ml of degassed Tyrode buffer B (flow rate, 2 ml/min). The column is closed, and sample is eluted with degassed Tyrode buffer B (flow rate, 2 ml/min). The first platelets appear in the 18–20 min fractions and are collected thereafter for 10 min in a closed plastic cup. GFP are reconstituted at a density of 4×108 platelets/ml with Tyrode buffer B and maintained at room temperature until use.

2.8.2.2. Experimental Course

Each concentration of drug is tested in triplicate using No. 72708 Sarstedt tubes. The total volume of each test sample is 500 μl. The concentration of 125 I-fibrinogen is constant for all samples (10 μg/500 μl).

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Competition reactions are carried out using a negative control (i.e., double distilled water), non-labeled fibrinogen, and increasing concentrations of test compound, as follows: • 100 μl 125 I-fibrinogen • 100 μl non-labeled fibrinogen or test drug (increasing concentrations of 10−10 to 10−3 M) • 50 μl ADP

2.8.2.4. Non-Specific Binding

Non-specific binding of 125 I-fibrinogen is defined as the level of binding of radioligand in the presence of 10−5 M non-labeled fibrinogen. The binding reaction is initiated by the addition of 250 μl of GFP (4×108 platelets/ml). The samples are incubated for 30 min at room temperature, and then 100 μl is transferred to a microcentrifuge tube containing 400 μl of a glucose solution. The tubes are centrifuged at 11,750 rpm for 2 min to separate bound 125 I-fibrinogen from free radioligand. The supernatant is carefully decanted and allowed to evaporate for approximately 30 min. The radioactivity of the platelet pellet is measured for 1 min using a gamma counter (efficiency in the range of 65.3%). Readers are referred to several excellent references for a detailed methodology and evaluation of various mechanisms and agents assessed using the fibrinogen binding assay (23, 24, 33, 34).

2.9. Euglobulin Clot Lysis Time 2.9.1. Purpose and Rational

Euglobulin lysis time is used as an indicator of the influence of compounds on fibrinolytic activity in rat blood and is based on the procedure of Gallimore et al. (35). The euglobulin fraction of plasma is separated from inhibitors of fibrinolysis by acid precipitation and centrifugation. Euglobulin predominantly consists of plasmin, plasminogen, plasminogen activator, and fibrinogen. Addition of thrombin to this fraction results in the formation of fibrin clots. The lysis time of these clots is related to the activity of activators of fibrinolysis (e.g., plasminogen activators). Thus, compounds that stimulate the release of t-PA from the vessel wall can be detected with this assay.

2.9.2. Procedure

Rats are anesthetized by ip injection of pentobarbital sodium (60 mg/kg) and placed on a heating pad (37◦ C). At the same time, the test solution, or vehicle as a control, is administered iv or ip. Twenty-five minutes later, the animals receive another ip injection of sodium pentobarbital (12 mg/kg) to maintain them in deep narcosis for 45 min.

2.9.2.1. Plasma Preparation

After the test compound is absorbed, the inferior caval vein is exposed by a midline excision and blood (1.8 ml) is drawn using a plastic syringe containing 0.2 ml of a 3.8% sodium citrate

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solution. The sample is thoroughly mixed, transferred to a plastic tube, and immediately immersed in ice. Plasma is obtained by centrifugation at 2,000×g for 10 min at 2◦ C. 2.9.2.2. Euglobulin Preparation

A 0.5 ml aliquot of plasma is added to 9.5 ml of ice-cold distilled water, and then the pH is brought to 5.3 by the addition of 0.13 ml of 1% acetic acid. The diluted plasma is kept on ice for 10 min and then the precipitated euglobulin fraction is collected by centrifugation at 2,000×g for 10 min at 2◦ C. The supernatant is discarded and the remaining fluid is removed by wicking away excess moisture with filter paper for 1 min. The euglobulin precipitate is dissolved in 1 ml of 0.12 M sodium acetate.

2.9.2.3. Euglobulin Lysis Assay

Aliquots (0.45 ml) of the euglobulin solution are transferred to test tubes, and 0.05 ml of thrombin (Test Thrombin, Behringwerke) (25 U/ml) is added. The tubes are transferred to a water bath at 37◦ C. The time interval between the addition of thrombin and complete lysis of the clots is recorded.

2.10. Flow Behavior of Erythrocytes 2.10.1. Purpose and Rational

The deformation of erythrocytes is an important rheological phenomenon in blood circulation (36). It allows the passage of normal red cells through capillaries with diameters smaller than that of the discoid cells and reduces the bulk viscosity of blood flowing in large vessels. In this test, the initial flow of filtrate is used as a measure of erythrocyte deformability. Prolonged filtration time can be attributed to two fundamental pathological phenomena: an increased rigidity of individual red cells and an increased tendency of the cells to aggregate. To simulate decreased red blood cell deformability, the erythrocytes are artificially modified by one (or a combination) of the following stress factors: • addition of calcium ions (increase in erythrocyte rigidity) • addition of lactic acid (decrease in pH value) • addition of 350–400 mmol NaCl (hyper-osmolarity) • storing the sample for at least 4 h (cellular ageing, depletion of ADP) This test is typically used to evaluate the effect of test compounds on the flow behavior of erythrocytes.

2.10.2. Procedure

Apparatus. Erythrocyte filtrometer model MF 4 (Fa. Myrenne, 52159 Roetgen, Germany) equipped with a membrane filter (Nuclepore Corp., Pleasanton, CA, USA) (pore diameter, 5– 10 μm; pore density, 4×105 pores/cm2 ). Ex vivo. Blood is collected from Beagle dogs (12–20 kg), rabbits (1.2–2.5 kg) or Wistar rats (150–300 g). Animals receive the test compound by oral, sc or iv administration 15, 60, 90, or 180 min before the withdrawal of blood.

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In vitro. Following the addition of test compound, blood is incubated at 37◦ C for 5 or 30 min. Freshly collected venous blood is anticoagulated with K-EDTA (1 mg/ml) or heparin (5 IU/ml heparin sodium) and then subjected to centrifugation at 3,000 rpm for 7 min. The supernatant (plasma) and buffy coat are removed and discarded. The packed erythrocytes are resuspended in autologous plasma containing 0.25% human albumin, and the haematocrit is adjusted to 10%. Red blood cells are then subjected to one of a combination of stress factors mentioned above. A portion (2 ml) of the stressed suspension is applied to the filtrometer and initial flow rate is determined. The filtration curve is plotted automatically. 2.11. Filterability of Erythrocytes 2.11.1. Purpose and Rational

The single erythrocyte rigidometer (SER) measures the deformability of individual red blood cells by measuring passage time through a pore under constant shear stress. In this assay, the passage times of single erythrocytes through one pore in a synthetic membrane are determined (37–39). The pore in the membrane essentially represents a capillary with a defined diameter and length. The driving pressure is produced by constant shear stress. The passage of red blood cells is measured based on changes in electrical current. For example, a constant current of 50–200 nA is applied, and passage of an erythrocyte through the pore is recorded as an interruption in current. This test is used to detect compounds that improve the filterability, and thus deformability, of erythrocytes. To simulate decreased red blood cell deformability, the erythrocytes are artificially modified by one or a combination of the following stress factors: • calcium ions (increase erythrocyte rigidity) • lactic acid (decreases pH value) • 350–400 mmol NaCl (generates a state of hyper-osmolarity) • storage for at least 4 h (cellular ageing and depletion of ADP)

2.11.2. Procedure

Apparatus. Single erythrocyte rigidometer (Myrenne, 52159 Roetgen, Germany). Ex vivo. Blood is collected from Beagle dogs (12–20 kg), rabbits (1.2–2.5 kg), or Wistar rats (150–300 g), or from human. The subject receives the test compound by oral, sc, or iv administration 15, 60, 90, or 180 min before the withdrawal of blood. In vitro. Following the addition of the test compound, blood samples are incubated at 37ºC for 5 or 30 min. Blood samples are mixed with K-EDTA (1 mg/ml blood) or heparin (5 IE/ml heparin sodium) to prevent clotting and then subjected to centrifugation at 3,000 rpm for 7 min. The plasma

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and buffy coat are removed and discarded. The packed erythrocytes are re-suspended in filtered HEPES buffer containing 0.25% human albumin and the haematocrit value is adjusted to <1%. The red blood cells are altered by one or a combination of stress factors, and then a portion (2 ml) of the stressed suspension is applied to the rigidometer. The passage time of a population of 250 erythrocytes (tm) is recorded. Cells that remain in the pore for more than 100 ms (tm > 100 ms) cause rheological occlusion. Untreated red blood cells serve as the control for this assay. 2.12. Erythrocyte Aggregation 2.12.1. Purpose and Rational

The aggregation of red blood cells into rouleaux and the transition of rouleaux into three-dimensional cell networks is a rheological parameter that decisively influences the flow behavior of blood, especially in disturbed microcirculation. In this test, an erythrocyte aggregometer is used to measure erythrocyte aggregation. The transparent measuring chamber in a cone-and-plate configuration is transilluminated by light of a defined wave length, and the intensity of the transmitted light, which is modified by the aggregation process, is recorded. The method has been used successfully to determine the effect of test compounds on erythrocyte aggregation (37, 40). Apparatus. SER (Fa. Myrenne, 52159 Roetgen, Germany). Ex vivo. Blood is collected from Beagle dogs (12–20 kg), rabbits (1.2–2.5 kg), or Wistar rats (150–300 g). The animals receive test compound by oral, sc, or iv administration 15, 60, 90, or 180 min before the withdrawal of blood. In vitro. Following the addition of test compound, the blood sample is incubated at 37◦ C for 5 or 30 min. Blood is obtained by venipuncture and mixed with K-EDTA (1 mg/ml) or heparin (5 IU/ml heparin sodium) to prevent clotting. Erythrocyte aggregation is determined in whole blood with a haematocrit of 40%. A portion (40 μl) of the blood is transferred to the measuring device and the red cells are dispersed at a shear rate of 600/s. After 20 s, flow is switched to stasis, and the extent of erythrocyte aggregation is determined photometrically.

3. In Vitro Models of Thrombosis A variety of methods have been used to assess the ex vivo and/or in vitro efficacy of platelet antagonists, including photometric aggregometry, whole blood electrical aggregometry, and particle counting, as described earlier. In photometric aggregometry, a sample is placed in a stirred cuvette in the optical light path

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between a light source and a light detector. Aggregate formation is monitored by a decrease in turbidity, and the extent of aggregation is measured as percent maximal light transmission. The major disadvantage of this technique is that it cannot be applied to whole blood, since the presence of erythrocytes interferes with optical detection. Furthermore, it is insensitive to the formation of small aggregates. Particle counters are used to quantitate the size and the number of particles suspended in an electrolyte solution by monitoring the electrical current between two electrodes immersed in the solution. Aggregation in this system is quantitated by counting the number of platelets before and after stimulation and is usually expressed as a percentage of the initial count (41). The disadvantage of this technique is that it cannot distinguish platelets and platelet aggregates from other blood cells of the same size. Thus, one is limited to counting only a fraction of single platelets as well as aggregates that are much larger than erythrocytes and leukocytes. Electrical aggregometry allows the detection of platelet aggregates as they attach to electrodes immersed in a stirred cuvette containing whole blood or a platelet suspension. Attachment results in a decrease in conductance between the two electrodes, which can be quantitated in units of electrical resistance. A disadvantage of this method is that it is not sensitive enough to detect small aggregates (42). This section will discuss two complementary in vitro flow models of thrombosis that can be used to accurately quantify platelet aggregation in anticoagulated whole blood and evaluate the inhibitory effect of platelet antagonists: (1) a viscometric flow cytometric assay that measures direct shear-induced platelet aggregation in the bulk phase (43) and (2) a parallel-plate perfusion chamber assay coupled with a computerized videomicroscopy system that quantifies the adhesion and subsequent aggregation of human platelets in anticoagulated whole blood flowing over an immobilized substrate (i.e., collagen I) (43, 44). We also discuss a third in vitro flow assay described by Mousa et al. (44) in which surface-anchored platelets are pre-incubated with a GPIIb/IIIa antagonist, unbound drug is washed away, and then THP-1 monocytic cells are perfused into the system, enabling the characterization of agents with markedly distinct affinities and receptor-bound lifetimes. 3.1. Cone-and-Plate Viscometry Under Shear-Flow Cytometry 3.1.1. Purpose and Rational

The cone-and-plate viscometer is an in vitro flow model used to investigate the effects of bulk fluid shear stress on suspended cells. Anticoagulated whole blood (or isolated cell suspension) is placed

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between the two stainless steel plates of the viscometer. Rotation of the upper conical plate causes a well-defined and uniform shear stress to be applied to the entire fluid medium (1). The shear rate (γ ) in this system can be readily calculated from the cone angle and the speed of the cone using the following formula: 2πω γ = , 60θcp where γ is the shear rate per second, ω is the cone rotational rate in revolutions per minute (rev/min) and θ cp is the cone angle in radians. The cone angle is typically in the range of 0.3–1.0◦ . Shear stress, τ , is proportional to shear rate, γ , as shown by τ = μγ , where μ is the viscosity of the cell suspension (the viscosity of anticoagulated whole blood is approximately 0.04 cp at 37◦ C). Rotational viscometers are capable of generating shear stress in the range of ∼2 dyn/cm2 (venous level) to greater than 200 dyn/cm2 (stenotic arteries). 3.1.2. Procedure

Single platelets and platelet aggregates generated in blood upon shear exposure are differentiated from other blood cells by their characteristic forward-scatter and fluorescence profiles from flow cytometric analysis using fluorophore-conjugated platelet-specific antibodies (43). This technique requires no washing or centrifugation steps that could potentially induce artifacts due to platelet activation and enables the analysis of platelet function in the presence of other blood elements.

3.1.2.1. Isolation of Human Platelets

Venous blood is drawn by venipuncture into polypropylene syringes containing either sodium citrate (0.38% final concentration) or heparin (10 U/ml final concentration). Anticoagulated whole blood is subjected to centrifugation at 160×g for 15 min to obtain PRP.

3.1.2.2. Isolation of WP

PRP is subjected to centrifugation again at 1,100×g for 15 min in the presence of 2 μM PGE1. The platelet pellet is re-suspended in HEPES-Tyrode buffer containing 5 mM EGTA and 2 μM PGE1. Platelets are washed and collected by centrifugation (1,100×g for 10 min) and then re-suspended in HEPES-Tyrode buffer at a cell density of 2×108 cells/ml and maintained at room temperature for no longer than 4 h before use in aggregation/adhesion assays.

3.1.2.3. Experimental Course

The steps described in this section outline the procedure used to quantify platelet aggregation induced by shear stress in the bulk phase as well as the inhibitory effects of platelet antagonists. For a detailed description, see Konstantopolous et al. (43).

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1. Incubate anticoagulated whole blood with platelet antagonist, or vehicle as a control, at 37◦ C for 10 min. 2. Place the sample (typically 500 μl) on the stationary plate of a cone-and-plate viscometer maintained at 37◦ C. 3. Remove a small aliquot (∼3 μl) from the pre-sheared blood sample, incubate in 1% formaldehyde in D-PBS (30 μl), and process as outlined below for sheared samples. 4. Expose the sample to well-defined shear levels (typically 4,000 s-1 in the absence of a platelet antagonist to induce significant platelet aggregation) in the presence or absence of a platelet antagonist for prescribed periods of time (typically 30–60 s). 5. Remove a small aliquot (∼3 μl) from the sheared sample and incubate immediately in 1% formaldehyde in D-PBS (30 μl). 6. Incubate the fixed blood samples (pre-sheared and sheared) with a saturating concentration of a fluorescent-labeled platelet-specific antibody, such as anti-GPIb (6D1)-FITC, for 30 min in the dark. 7. Dilute specimens with 2 ml of 1% formaldehyde, and analyze by flow cytometry 8. Flow cytometric analysis is used to distinguish platelets from other blood cells on the basis of their characteristic forwardscatter and fluorescence profiles (Fig. 1.1). Data acquisition is carried out on each sample for a defined period (usually 100 s), allowing equal volumes for the pre-sheared and sheared specimens to be achieved. Percent platelet aggregation is determined based on the disappearance of single platelets and increase in platelet aggregates using the following formula: % platelet aggregation = (1 − Ns /Nc × 100) where Ns represent the single platelet population of the sheared specimen and Nc represents the single platelet population of the pre-sheared specimen. By comparing the extent of platelet aggregation in the presence and absence of a platelet antagonist, antiplatelet effects can be readily determined. 3.2. Platelet Adhesion and Aggregation Under Dynamic Shear 3.2.1. Purpose and Rational

This section describes an in vitro flow model of platelet thrombus formation that can be used to evaluate the ex vivo and/or in vitro efficacy of platelet antagonists. Thrombus formation can be initiated by platelet adhesion from rapidly flowing blood

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Fig. 1.1. Quantification of shear-induced platelet aggregation by flow cytometry. Panel A corresponds to an un-sheared blood specimen. Panel B corresponds to a blood specimen that has been subjected to a pathologically high level of shear stress for 30 s. As can be seen in the Figure there are three distinct cell populations. The upper population consists of platelets and platelet aggregates. The “red blood cell–platelet” population corresponds to platelets associated with erythrocytes and leukocytes (see Evaluation, Comment 4). The white blood cell population consists of some leukocytes that have elevated levels of FITC autofluorescence. The left vertical line separates single platelets (≤ 4.5 μm in diameter) from platelet aggregates, whereas the right vertical line separates “small” from “large” platelet aggregates. The latter were defined to be larger than 10 μm in equivalent sphere diameter.

onto exposed sub-endothelial surfaces of injured vessels presenting collagen and vWF, resulting in platelet activation and aggregation. Konstantopolous et al. (43) described the use of a parallel-plate flow chamber that provides a controlled and welldefined flow environment (i.e., chamber geometry and flow rate through the chamber). Wall shear stress, τ w , assuming a Newtonian and incompressible fluid, can be calculated using the following formula: 6μQ τw = , wh 2 where Q is the volumetric flow rate, μ is the viscosity of the flowing fluid, h is the channel height, and w is the channel width.

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A typical flow chamber consists of a transparent polycarbonate block, a gasket whose thickness determines the channel depth, and a glass coverslip coated with an extracellular matrix protein, such as type I fibrillar collagen. The apparatus is held together by vacuum. Shear stress is generated by flowing fluid (i.e., anticoagulated whole blood or isolated cell suspensions) through the chamber over the immobilized substrate under controlled kinematic conditions using a syringe pump. Recently, Mousa et al. (44) combined the parallel-plate flow chamber with a computerized epifluorescence videomicroscopy system, enabling separation and real-time visualization of adhesion and subsequent aggregation of human platelets in anticoagulated whole blood (or isolated platelet suspensions) flowing over an immobilized substrate. 3.2.2. Procedure 3.2.2.1. Preparation of Collagen-Coated Surfaces

1. Dissolve 500 mg of collagen type I from bovine achilles tendon into 200 ml of 0.5 M acetic acid, pH 2.8. 2. Homogenize for 3 h. 3. Centrifuge the homogenate at 200×g for 10 min, collect the supernatant, and then measure collagen concentration by modified Lowry analysis. 4. Coat glass coverslips with 200 μl of fibrillar collagen I suspension so that all but the first 10 mm of the slide is covered (coated area = 12.7 mm×23 mm) and then place coated coverslips in a humid environment at 37◦ C for 45 min. 5. Rinse the slides to remove excess collagen with 10 ml of warm (37◦ C) D-PBS and then assembly the flow chamber.

3.2.2.2. Platelet Perfusion

1. Add the fluorescent dye quinacrine dihydrochloride to anticoagulated whole blood samples at a final concentration of 10 μM immediately after blood collection. 2. Prior to the perfusion experiment, incubate blood with platelet antagonist, or vehicle as a control, at 37◦ C for 10 min. 3. Perfuse anticoagulated whole blood through the flow chamber for 1 min at wall shear rates of 100/s (typical of venous circulation) to 1,500/s (representative of partially constricted arteries) for prescribed periods of time (i.e., 1 min). Platelet–substrate interactions are monitored in real time using an inverted microscope equipped with an epifluorescence illumination apparatus and a silicon-intensified target video camera and recorded on videotape. The microscope stage and flow chamber are maintained at 37◦ C by a heating module and incubator enclosure during the experiment.

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4. Videotaped images are digitized and computer analyzed at 5, 15, and 60 s for each perfusion experiment. The number of adherent individual platelets in the microscopic field of view during the initial 15 s of flow is determined by image processing and used as the measurement of platelet adhesion that initiates platelet thrombus formation. The number of platelets in each individual thrombus is calculated as the total thrombus intensity (area×fluorescence intensity) divided by the average intensity of single platelets determined in the 5-s images. By comparing the extents of platelet aggregation in the presence and absence of a platelet antagonist, antiplatelet efficacy can be determined (Fig. 1.2). Along these lines, any potential inhibitory effects of a platelet antagonist on platelet adhesion can be readily assessed.

Fig. 1.2. Three-dimensional computer-generated representation of platelet adhesion and subsequent aggregation on collagen I/von Willebrand factor from normal heparinized blood perfused in the absence (control) or presence of a GPIIb/IIIa antagonist (XV454) at 37◦ C for 1 min at 1,500/s.

3.3. Cell Adhesion to Immobilized Platelets: Parallel-Plate Flow Chamber 3.3.1. Purpose and Rational

This section outlines an in vitro flow assay to distinguish agents with markedly distinct affinities and off-rates. In this assay, immobilized platelets are pretreated with a GPIIb/IIIa antagonist, and any unbound drug is washed away before the perfusion of monocytic THP-1 cells. Using this technique, Albulencia et al. (45) demonstrated that agents with slow platelet off-rates, such as XV454 (t1/2 of dissociation = 110 min; Kd = 1 nM) and abciximab (t1/2 of dissociation = 40 min; Kd = 9.0 nM), which are present predominantly as receptor-bound entities in plasma with little unbound agent, can effectively block platelet heterotypic interactions. In contrast, agents with relatively fast

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platelet dissociation rates, such as orbofiban (t1/2 of dissociation = 0.2 min; Kd >110 nM), the antiplatelet efficacy of which depends on the plasma concentration of the active drug, do not exhibit such inhibitory effects (44). 3.3.2. Procedure 3.3.2.1. Preparation of 3-Aminopropyltriethoxysilane (APES)-Treated Glass Slides

1. Soak glass coverslips overnight in 70% nitric acid. 2. Wash coverslips with tap water for 4 h. 3. Dry coverslips by washing once with acetone, and then immerse in a 4% solution of APES in acetone for 2 min. 4. Repeat step 3, followed by a final rinse of the glass coverslips with acetone. 5. Wash coverslips 3 times with water and dry overnight.

3.3.2.2. Immobilization of Platelets on APES-Treated Glass Slides

1. Layer WP or PRP (2×108 cells/ml) on the surface of an APES-coated coverslip at a density of approximately 30 μl/cm2 .

3.3.2.3. Monocytic THP-1 Cell–Platelet Adhesion Assay

1. Assemble the platelet-coated coverslip in a parallel-plate flow chamber. Mount the chamber on the stage of an inverted microscope equipped with a CCD camera connected to a VCR and TV monitor.

2. Allow platelets to bind to the coverslip in a humid environment at 37◦ C for 30 min.

2. Perfuse antiplatelet antagonist at the desired concentration, or vehicle as a control, over surface-bound platelets and incubate for 10 min. The extent of platelet activation can be further modulated by chemical agonists such as thrombin (0.02–2 U/ml) during the 10-min incubation period. The microscope stage and flow chamber are maintained at 37◦ C with a heating module and incubator enclosure during the experiment. 3. In some experiments, unbound platelet antagonist is removed by a brief washing step (4 min) prior to the perfusion of the cells of interest over the platelet layer. Alternatively, platelet antagonist at the desired concentration is continuously maintained in the perfusion buffer during the entire course of the experiment. 4. Perfuse cells (i.e., THP-1 monocytic cells, leukocytes, tumor cells, protein-coated beads) over surface-bound platelets in the presence or absence of platelet antagonist (see above) at the desired flow rate for prescribed periods of time. Cell binding to immobilized platelets is monitored in real time and recorded on videotape. 5. Determine the extent of cell tethering, rolling, and stationary adhesion to immobilized platelets, as well as the average

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velocity of rolling cells. Comparison of the extent of cell tethering, rolling, and stationary adhesion to immobilized platelets in the presence and absence of platelet antagonist (Fig. 1.3) will provide a measure of antiplatelet activity.

Fig. 1.3. Phase-contrast photomicrograph of THP-1 cells (phase bright objects) attached to a layer of thrombin-treated platelets (phase dark objects) after THP-1 cell perfusion for 3 min at a shear stress level of 1.5 dyn/cm2 .

3.3.3. Evaluation

1. Low-speed centrifugation of whole blood results in the separation of platelets (top layer) from larger and denser cells such as leukocytes and erythrocytes (bottom layer). To minimize leukocyte contamination of PRP, slowly aspirate the uppermost 2/3 of the platelet layer after centrifugation. Also, certain rare platelet disorders such as Bernard–Soulier syndrome (BSS) are characterized by larger than normal platelets which must be isolated by allowing whole blood to separate by gravity for 2-h post-venipuncture. 2. The mechanical force most relevant to platelet-mediated thrombosis is shear stress. Normal time-averaged levels of venous and arterial shear stress range from 1–5 dyn/cm2 to 6–40 dyn/cm2 , respectively. However, fluid shear stress may reach levels well over 200 dyn/cm2 in small arteries and arterioles that are partially obstructed by atherosclerotic lesions or vascular spasm. The cone-and-plate viscometer and parallel-plate flow chamber are two of the most common devices used to simulate fluid mechanical shear stress in blood vessels. 3. Due to the high concentration of platelets and erythrocytes in whole blood, small aliquots (3 μl) of pre-sheared and post-sheared samples must be obtained and processed prior to the flow cytometric analysis. This will minimize artifacts

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produced when platelets and erythrocytes pass through the light beam of a flow cytometer at the same time. 4. The “red blood cell–platelet” population in the flow cytometry histogram of Fig. 1.1 represents 3–5% of the displayed cells. A small fraction (5%) of this population likely represents leukocyte–platelet aggregates, based on analysis using an anti-CD45 monoclonal antibody. The remaining events correspond to platelet-associated erythrocytes. However, there is evidence that the majority of the latter population is an artifact generated by the simultaneous passage of a platelet and an erythrocyte through the light beam of the flow cytometer. That this population represents an artifact is supported by the observation that further dilution of pre-sheared and sheared blood specimens and/or reduction of the sample flow rate during the flow cytometric analysis results in a dramatic decrease in the “red blood cell–platelet” population. 5. Collagen density on the glass coverslip after rinsing with D-PBS can be determined by measuring the difference in average weight of 20 clean uncoated slides and 20 collagentreated slides. 6. Experiments are optimally monitored approximately 100– 200 μm downstream of the collagen/glass interface using a 60× FLUOR objective and a 1× projection lens, which gives a 3.2×104 μm2 field of view. Monitoring closer to the interface may yield non-reproducible results due to variations in the collagen layering in that region. Positions farther downstream are to be avoided as well so as to minimize the effects of upstream platelet adhesion and subsequent aggregation on the fluid dynamic environment and bulk platelet concentration. 7. The fluorescence intensity emitted by a single platelet can be determined by subtracting a digitized background image taken at the onset of perfusion, prior to platelet adhesion to the collagen I surface, from a subsequent image acquired 5 s after the initiation of platelet adhesion. The fluorescence intensity of a single platelet is represented by the mean gray level (black = 0; white = 255) of the platelet, obtained using image processing software (i.e., OPTIMAS; Agris-Schoen Vision Systems, Alexandria, VA, USA), multiplied by the corresponding area (total number of pixels) covered by the platelet. The intensity values for all single platelet events are averaged at the 5-s time point to arrive at an average single platelet intensity. 8. A single field of view (10×; 0.55 mm2 ) is monitored during the 3-min period of the experiment. At the conclusion

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of the experiment, five additional fields of view (0.55 mm2 ) are monitored for 15 s each. The following parameters can be quantified: (a) the total number of interacting cells per mm2 during the 3-min perfusion experiment; (b) the number of stationary interacting cells per mm2 after 3 min of shear flow; (c) the percentage of total interacting cells that are stationary after 3 min of shear flow; and (d) the average rolling velocity (μm/s) of interacting cells. The number of interacting cells/mm2 is determined manually by reviewing the videotapes. Stationary interacting cells/mm2 are those cells that move <1-cell radius within 10 s of the conclusion of the 3-min perfusion. To quantify cell numbers, images can be digitized from the videotape recorder using an imaging software package (e.g., OPTIMAS). Rolling velocities can be computed using image processing as the distance traveled by the centroid of the rolling cell divided by the time interval. References 1. Konstantopoulos, K., Kukreti, S., and McIntire, L.V. (1998) Biomechanics of cell interactions in shear fields Adv drug delivery rev 33, 141–64. 2. Alevriadou, B.R., Moake, J.L., Turner, N.A., Ruggeri, Z.M., Folie, B.J., Phillips, M.D., Schreiber, A.B., Hrinda, M.E., and McIntire, L.V. (1993) Real-time analysis of shear-dependent thrombus formation and its blockade by inhibitors of von Willebrand factor binding to platelets Blood 81, 1263–76. 3. Turitto, V.T. (1982) Blood viscosity, mass transport, and thrombogenesis Prog Hemost Thromb 6, 139–77. 4. Palmer, R.L. (1984) Laboratory diagnosis of bleeding disorders. Basic screening tests Postgraduate Med 76, 137–42, 47–8. 5. Nilsson, I. Assessment of blood coagulation and general haemostasis. In: Bloom, A.L, and Thomas, D.P., eds. Haemostasis and Thrombosis, 2nd edition. Harlow: Longman Group (UK) Limited;1987:922–32 6. Hartert, H. (1948) Blutgerinnungsstudien mit der Thrombelastographie, einem neuen Untersuchungsverfahren Klinische Wochenschrift 26, 577–83. 7. Khurana, S., Mattson, J.C., Westley, S., O’Neill, W.W., Timmis, G.C., and Safian, R.D. (1997) Monitoring platelet glycoprotein IIb/IIIa-fibrin interaction with tissue factor-activated thromboelastography J Lab Clin Med 130, 401–11. 8. Mousa, S., Khurana, S., and Forsythe, M. (2000) Comparative in vitro efficacy of dif-

9.

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ferent platelet glycoprotein IIb/IIIa antagonists on platelet-mediated clot strength induced by tissue factor with use of thromboelastography: differentiation among glycoprotein IIb/IIIa antagonists Arterioscler Thromb Vasc Biol 20, 1162–7. Bhargava, A.S., Freihube, G., and Gunzel, P. (1980) Characterization of a new potent heparin. 3rd Communication: determinations of anticoagulant activity of a new potent heparin preparation by thrombelastography in vitro using citrated dog and human blood Arzneimittel-Forschung 30, 1256–8. Barabas, E., Szell, E., and Bajusz, S. (1993) Screening for fibrinolysis inhibitory effect of synthetic thrombin inhibitors Blood Coagul Fibrinol 4, 243–8. Scherer, R.U., Giebler, R.M., Schmidt, U., Paar, D., Wust, T., Spangenberg, P., Militzer, K., Hirche, H., and Kox, W.J. (1995) Shorttime rabbit model of endotoxin-induced hypercoagulability Lab Animal Sci 45, 538–46. Mousa, S.A., Bozarth, J.M., Seiffert, D., and Feuerstein, G.Z. (2005) Using thrombelastography to determine the efficacy of the platelet glycoprotein IIb/IIIa antagonist, roxifiban, on platelet/fibrin-mediated clot dynamics in humans Blood Coagul Fibrinol 16, 165–71. Zuckerman, L., Cohen, E., Vagher, J.P., Woodward, E., and Caprini, J.A. (1981) Comparison of thrombelastography with

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common coagulation tests Thromb Haemost 46, 752–6. Chandler, A.B. (1958) In vitro thrombotic coagulation of the blood; a method for producing a thrombus Lab Invest; J Tech Methods Pathol 7:110–4. Robbie, L.A., Young, S.P., Bennett, B., and Booth, N.A. (1997) Thrombi formed in a Chandler loop mimic human arterial thrombi in structure and RAI-1 content and distribution Thromb Haemost 77, 510–5. Stringer, H.A., van Swieten, P., Heijnen, H.F., Sixma, J.J., and Pannekoek, H. (1994) Plasminogen activator inhibitor-1 released from activated platelets plays a key role in thrombolysis resistance. Studies with thrombi generated in the Chandler loop Arterioscler Thromb 14, 1452–8. van Giezen, J.J., Nerme, V., and Abrahamsson, T. (1998) PAI-1 inhibition enhances the lysis of the platelet-rich part of arterial-like thrombi formed in vitro. A comparative study using thrombi prepared from rat and human blood Blood Coagul Fibrinol 9, 11–8. Born, G. (1962) Quantitative investigations into the aggregation of blood platelets J Physiol (London) 162, 67P–8P. Born, G.V. (1962) Aggregation of blood platelets by adenosine diphosphate and its reversal Nature 194, 927–9. Haskel, E.J., and Abendschein, D.R. (1989) Deaggregation of human platelets in vitro by an RGD analog antagonist of platelet glycoprotein IIb/IIIa receptors Thromb Res 56, 687–95. Breddin, K., Grun, H., Krzywanek, H.J., and Schremmer, W.P. (1975) Measurement of spontaneous platelet aggregation. Platelet aggregation test III (author’s transl) Klinische Wochenschrift 53, 81–9. Klose, H.J., Rieger, H., and SchmidSchonbein, H. (1975) A rheological method for the quantification of platelet aggregation (PA) in vitro and its kinetics under defined flow conditions Thromb Res 7, 261–72. Marguerie, G.A., Edgington, T.S., and Plow, E.F. (1980) Interaction of fibrinogen with its platelet receptor as part of a multistep reaction in ADP-induced platelet aggregation J Biol Chem 255, 154–61. Marguerie, G.A., Plow, E.F., and Edgington, T.S. (1979) Human platelets possess an inducible and saturable receptor specific for fibrinogen J Biol Chem 254, 5357–63. Lumley, P., and Humphrey, P.P. (1981) A method for quantitating platelet aggregation and analyzing drug-receptor interactions on platelets in whole blood in vitro J Pharmacol Methods 6, 153–66.

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26. Fratantoni, J.C., and Poindexter, B.J. (1990) Measuring platelet aggregation with microplate reader. A new technical approach to platelet aggregation studies Am J Clin Pathol 94, 613–7. 27. Ammit, A.J., and O’Neill, C. (1991) Rapid and selective measurement of plateletactivating factor using a quantitative bioassay of platelet aggregation J Pharmacol Methods 26, 7–21. 28. Markell, M.S., Fernandez, J., Naik, U.P., Ehrlich, Y., and Kornecki, E. (1993) Effects of cyclosporine-A and cyclosporineG on ADP-stimulated aggregation of human platelets Ann N Y Acad Sci 696, 404–7. 29. Cardinal, D.C., and Flower, R.J. (1980) The electronic aggregometer: a novel device for assessing platelet behavior in blood J Pharmacol Methods 3, 135–58. 30. Tohgi, H., Takahashi, H., Watanabe, K., Kuki, H., and Shirasawa, Y. (1996) Development of large platelet aggregates from small aggregates as determined by laser-light scattering: effects of aggregant concentration and antiplatelet medication Thromb Haemost 75, 838–43. 31. Matsuo, T., Koide, M., Sakuramoto, H., Matsuo, M., and Fuji, K. (1997) Small size of platelets aggregates detected by light scattering in young habitual smokers Thromb Haemost 71(suppl), 71. 32. Xiao, Z., and Theroux, P. (1998) Platelet activation with unfractionated heparin at therapeutic concentrations and comparisons with a low-molecular-weight heparin and with a direct thrombin inhibitor Circulation 97, 251–6. 33. Bennett, J.S., and Vilaire, G. (1979) Exposure of platelet fibrinogen receptors by ADP and epinephrine J Clin Invest 64, 1393–401. 34. Mendelsohn, M.E., O’Neill, S., George, D., and Loscalzo, J. (1990) Inhibition of fibrinogen binding to human platelets by Snitroso-N-acetylcysteine J Biol Chem 265, 19028–34. 35. Gallimore, M.J., Tyler, H.M., and Shaw, J.T. (1971) The measurement of fibrinolysis in the rat Thromb Diath Haemorrh 26, 295–310. 36. Teitel, P. (1977) Basic principles of the ‘Filterability test’ (FT) and analysis of erythrocyte flow behavior Blood Cells 3, 55–70. 37. Kiesewetter, H., Dauer, U., Teitel, P., Schmid-Schonbein, H., and Trapp, R. (1982) The single erythrocyte rigidometer (SER) as a reference for RBC deformability Biorheology 19, 737–53. 38. Roggenkamp, H.G., Jung, F., and Kiesewetter, H. (1983) A device for the electrical

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Mousa measurement of the deformability of red blood cells Biomedizinische Technik 28, 100–4. Seiffge, D., and Behr, S. (1986) Passage of red blood cells in the SER: their distribution and influences of various extrinsic and intrinsic factors Clin Hemorheol 6, 1510164. Schmid-Schonbein, H., von Gosen, J., Heinich, L., Klose, H.J., and Volger, E. (1973) A counter-rotating "rheoscope chamber" for the study of the microrheology of blood cell aggregation by microscopic observation and microphotometry Microvasc Res 6, 366–76. Jen, C.J., and McIntire, L.V. (1984) Characteristics of shear-induced aggregation in whole blood J Lab Clin Med 103, 115–24. Sweeney, J.D., Labuzzetta, J.W., Michielson, C.E., and Fitzpatrick, J.E. (1989) Whole blood aggregation using impedance and particle counter methods Am J Clin Pathol 92, 794–7.

43. Konstantopoulos, K., Kamat, S.G., Schafer, A.I., Banez, E.I., Jordan, R., Kleiman, N.S., and Hellums, J.D. (1995) Shear-induced platelet aggregation is inhibited by in vivo infusion of an anti-glycoprotein IIb/IIIa antibody fragment, c7E3 Fab, in patients undergoing coronary angioplasty Circulation 91, 1427–31. 44. Mousa, S.A., Abulencia, J.P., McCarty, O.J., Turner, N.A., and Konstantopoulos, K. (2002) Comparative efficacy between the glycoprotein IIb/IIIa antagonists roxifiban and orbofiban in inhibiting platelet responses in flow models of thrombosis J Cardiovasc Pharmacol 39, 552–60. 45. Abulencia, J.P., Tien, N., McCarty, O.J., Plymire, D., Mousa, S.A., and Konstantopoulos, K. (2001) Comparative antiplatelet efficacy of a novel, nonpeptide GPIIb/IIIa antagonist (XV454) and abciximab (c7E3) in flow models of thrombosis Arterioscl Thromb Vasc Biol 21, 149–56.

Chapter 2 In Vivo Models for the Evaluation of Antithrombotics and Thrombolytics Shaker A. Mousa Abstract The development and application of animal models of thrombosis have played a crucial role in the discovery and validation of novel drug targets and the selection of new agents for clinical evaluation, and have informed dosing and safety information for clinical trials. These models also provide valuable information about the mechanisms of action/interaction of new antithrombotic agents. Small and large animal models of thrombosis and their role in the discovery and development of novel agents are described. Methods and major issues regarding the use of animal models of thrombosis, such as positive controls, appropriate pharmacodynamic markers of activity, safety evaluation, species specificity, and pharmacokinetics, are highlighted. Finally, the use of genetic models of thrombosis/hemostasis and how these models have aided in the development of therapies that are presently being evaluated clinically are presented. Key words: Animal models, coronary thrombosis, antithrombotic agents, pharmacodynamics, Folts, Wessler, thrombocytopenia, genetic models.

1. Introduction The general understanding of the pathophysiology of thrombosis is based on the observations of Virchow in 1856. Three factors responsible for thrombogenesis are proposed including obstruction of blood flow, changes in the properties of blood constituents (hypercoagulability), and vessel wall injury. Experimental models of thrombosis focus on one, two, or all three factors of Virchow’s triad. Therefore, they differ with respect to the prothrombotic challenge, i.e., stenosis, stasis, vessel wall injury (mechanical, electrical, chemical, photochemical, laser-light), insertion of foreign S.A. Mousa (ed.), Anticoagulants, Antiplatelets, and Thrombolytics, Methods in Molecular Biology 663, DOI 10.1007/978-1-60761-803-4_2, © Springer Science+Business Media, LLC 2003, 2010

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surface, or injection of a prothrombotic factor, as well as vessel type and animal species. Roughly, two types of models can be differentiated (1): (1) models in which thrombi are produced in veins by stasis and/or injection of a procoagulant factor resulting in fibrin-rich “red” venous type thrombi and (2) models in which thrombi are produced in arteries by vessel wall injury and/or stenosis resulting in platelet-rich “white” mural thrombi. This distinction is not strict because platelets and the coagulation system influence each other. Drugs that prevent fibrin formation may well act in arterial models and vice versa. Thrombosis models are usually performed in healthy animals. The underlying chronic diseases in human, namely atherosclerosis or thrombophilias, are not addressed in the models. Thus, any model is limited with regard to clinical relevance. The pharmacological effectiveness of a new antithrombotic drug should be studied in more than one animal model. Despite these limitations, animal models predict the clinical effectiveness of drugs for the treatment and prevention of thrombotic diseases fairly well. A list of such drugs is presented in a recent review by Leadleey et al. (2). The clinical usefulness of an antithrombotic drug is determined in part by safety/efficacy ratio with respect to bleeding risk. Assessment of this parameter of the hemostatic system should therefore be included in the models if possible. The development of antithrombotic agents requires preclinical assessment of the biochemical and pharmacologic effects of these drugs. It is important to note that second- and thirdgeneration antithrombotic drugs are devoid of in vitro anticoagulant effects, yet in vivo, by virtue of endogenous interactions, these drugs produce potent antithrombotic actions. The initial belief that an antithrombotic drug must exhibit in vitro anticoagulant activity is no longer valid. This important scientific observation has been possible only because of the availability of animal models. Several animal models utilizing species such as rats, rabbits, dogs, pigs, and monkeys have been made available for routine use. Other animal species such as the hamster, mouse, cat, and guinea pig have also been used. Species variations are an important consideration in selecting a model and interpreting the results, as these variations can result in different antithrombotic effects. Rats and rabbits are the most commonly used species in which both arterial and venous thromboses have been investigated. Both pharmacologic and mechanical means have been used to produce a thrombogenic effect in these models. Both rat and rabbit models for studying bleeding effects of drugs have also been developed. The rabbit ear blood loss model is most commonly used to test the hemorrhagic effect of drugs. The rat tail bleeding models have also been utilized for the study of several antithrombotic drugs. These animal models have been well established and can be used for the development of antithrombotic drugs. It is also pos-

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sible to use the standardized bleeding and thrombosis models to predict the safety and efficacy of drugs. Thus, in addition to the evaluation of in vitro potency, the endogenous effects of antithrombotic drugs can be investigated. Such standardized methods can be recommended for inclusion in pharmacopoeial screening procedures. Numerous models have now been developed to mimic a variety of clinical conditions where antiplatelet and antithrombotic drugs are used, including myocardial infarction, stroke, cardiopulmonary bypass, trauma, peripheral vascular diseases, and restenosis. While dog and primate models are relatively expensive, they have also provided useful information on the pharmacokinetics and pharmacodynamics of antithrombotic drugs. The primate models in particular have been extremely useful, as the hemostatic pathways in these species are comparable to those in humans. The development of such agents as the specific glycoprotein IIb/IIIa inhibitor antibodies relies largely on these models. These models are of pivotal value in the development of antithrombotic drugs and provide extremely useful data on the safety and efficacy of new drugs developed for human use. In most animal models of thrombosis, healthy animals are challenged with thrombogenic (pathophysiologic) stimuli and/or physical stimuli to produce thrombotic or occlusive conditions. These models are useful for the screening of antithrombotic drugs. 1.1. Stasis Thrombosis Model

Since its introduction by Wessler in 1959 (3), the rabbit model of jugular stasis thrombosis has been extensively used for the pharmacologic screening of antithrombotic agents. This model has also been adapted for use in rats (4). In the stasis thrombosis model, a hypercoagulable state is mimicked by the administration of one of a number of thrombogenic challenges, including human serum (5), thromboplastin (6), activated prothrombin complex concentrates (7), factor Xa (8) and recombinant relipidated tissue factor (9). Administration of such agents produces a hypercoagulable state. Diminution of blood flow achieved by ligating the ends of vessel segments serves to augment the prothrombotic environment. The thrombogenic environment produced in this model simulates venous thrombosis where both the blood flow and the activation of coagulation play a role in the development of a thrombus.

1.2. Models Based on Vessel Wall Damage

The formation of a thrombus is not solely induced by a plasmatic hypercoagulable state. In the normal vasculature, the intact endothelium provides a non-thrombogenic surface over which the blood flows. Disruption of the endothelium exposes subendothelial tissue factor and collagen, which activate the coagulation and platelet aggregation processes, respectively. Endothelial damage can be induced experimentally by physical means

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(clamping, catheter), chemical means (fluorescein isothyocynate, Rose Bengal, ferrous chloride), thermal injury, or electrolytic injury. Each setting in the design of an animal model can answer specific question in relation to certain thrombotic disorders in human. However, the ultimate model of human thrombosis is in human. 1.3. Issues to be Taken in Consideration in Evaluating Antithrombotics 1.3.1. Effect on Hemostasis

Antithrombotic and anticoagulant drugs are effective in the control of thrombogenesis at various levels. These drugs are also capable of producing hemorrhagic effects that cannot be predicted using in vitro testing methods. The bleeding effects of a drug may be direct or indirect; hence assessing efficacy/safety ratios could be a useful parameter. Single versus repeated exposure. Repeated administration of drugs can result in a cumulative response that may alter the pharmacokinetic and pharmacodynamic indices of a given agent. It is only through the use of animal models that such information can be generated. Furthermore, since antithrombotic drugs represent a diverse class of agents, their interactions with physiologically active endogenous proteins can only be studied using animal models.

1.3.2. Choice of Species

Species variation plays an important role in thrombotic, hemostatic, and hemorrhagic responses. While there is no set formula to determine the relevance of the results obtained with animal models to man, the use of animal models can provide valuable information on the relative potency of drugs, their bioavailability after various routes of administration, and their pharmacokinetic behavior. Specific studies have provided data on the species relevance of the responses in different animal models to the projected human responses. Thus, the use of animal models in the evaluation of different drugs can provide useful data to compare different drugs within a class. However, caution must be exercised in extrapolating such results to the human clinical condition.

1.3.3. Selection of Animal Model

The selection of animal model for the evaluation of antithrombotic effects depends on several factors. The interaction of a given drug with the blood and vascular components and its metabolic transformation are important considerations. Thus, ex vivo analysis of blood along with the other endpoints can provide useful information on the effects of different drugs. Unlike the screening of drugs such as antibiotics, antithrombotic drugs require multi-

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parametric endpoint analysis. Animal models are the most useful system in the evaluation of the effects of these drugs. Finally, it should be stressed that the evaluation of pharmacopoeial and in vitro potency of antithrombotic drugs does not necessarily reflect the in vivo safety/efficacy profile. Endogenous modulation, such as the release of tissue factor pathway inhibitor (TFPI) by heparins, plays a very important role in the overall therapeutic index of many drugs. Such data can only be obtained using animal models. It is therefore important to design experiments where several data points can be obtained. This information is of crucial value in the assessment of antithrombotic drugs and cannot be substituted by other in vitro or tissue culture-based methods. Several excellent reviews covering the different theoretical and technical aspects of thrombosis and thrombolysis models have been published previously (2, 11–15). These reviews, along with more specialized reviews of models of atherosclerosis (16), restenosis (17), and stroke (18) provide comprehensive information regarding the details of many models and provide the pathological rationale for using specific models for specific diseases. In addition, the advantages and disadvantages of each model of thrombosis and thrombolysis are described in these reviews. This chapter focuses on the use of thrombosis models in the drug discovery process, with emphasis on the practical application of these models. Examples from studies evaluating therapeutic approaches that target various antithrombotic mechanisms will be presented to demonstrate the current use of thrombosis models in drug discovery. Important issues in evaluating novel antithrombotic compounds will also be addressed. In addition, evidence demonstrating the clinical relevance of preclinical data derived from animal models of thrombosis will also be presented. Finally, a summary of the use of genetic models of thrombosis/hemostasis and their current and potential use in drug discovery will also be discussed

2. Animal Models of Thrombosis 2.1. Stenosis- and Mechanical Injury-Induced Coronary Thrombosis (Folts Model) 2.1.1. Purpose and Rationale

Thrombosis in stenosed human coronary arteries is one of the most common thrombotic diseases leading to unstable angina,

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Fig. 2.1. Technique for monitoring platelet aggregation in the partially obstructed left circumflex coronary artery of the dog. Electromagnetic flow probes measure blood flow iii nil/mm. Partial obstruction of the coronary artery with a plastic Lexan cylinder results in episodic cyclical reductions in coronary blood flow that are due to platelet-dependent thrombus formation. Every 2–3 mm the thrombus must be mechanically shaken loose (SL) to restore blood.

acute myocardial infarction, or sudden death. Treatment with angioplasty, thrombolysis, or bypass grafts can expose new thrombogenic surfaces, and re-thrombosis may occur. The mechanisms responsible for this process include interactions of platelets with the damaged arterial wall and platelet aggregation. In 1976, Folts and co-workers (19) described a model of repetitive thrombus formation, or cyclic flow reductions (CFRs), in stenosed coronary arteries of open-chest, anesthetized dogs (Fig. 2.1). This model is also applicable to the rabbit femoral or carotid artery (20). Using this model, several groups have described the antithrombotic effects of a variety of drugs, primarily prostaglandin-inhibitors, prostacyclin-mimetics, or fibrinogen receptor antagonists (21–27). The combination of two thrombogenic stimuli leads to the development of CFRs in this model: severe, concentric stenosis and focal, intimal injury. With few exceptions, CFRs will not develop unless both stimuli exist.

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The rheological conditions required to produce turbulence and stasis upon vessel narrowing dictate that lumenal diameter be reduced by at least 50%. Two- to three-millimeter long constricR tors are cut from Lexan rods readily available from local plastics distributors. One center hole of varying diameter and two smaller collar holes are drilled, into which the prongs of snap-ring pliers fit to spread the constrictor’s slit in the top-central portion to apply or remove them. Other plastics will suffice, but Lexan is ideal because of its strength and resiliency. Both circumflex and left anterior descending (LAD) coronary arteries have been used in this model. Besides personal preferences, we know of no physiologic basis for preferring one to the other. Owing to the prominent auto-regulation of coronary circulation, it is difficult to assess the severity of a stenosis on the basis of changes in basal coronary blood flow (CBF). However, the robust reactive hyperemia (RH) characteristic of the coronary circulation provides a powerful tool with which to gauge the severity of the stenosis. With gradual narrowing, basal CBF will remain unchanged or decline negligibly, whereas RH will begin to decline sooner as the vasodilatory reserve of downstream vessels is progressively exhausted. Reduction of lumenal diameter to this degree is required if one wishes to produce CFRs in a high percentage (i.e., >90%) of dogs. It is also critical if one wishes to compare two or more drugs in this model and draw meaningful conclusions about drug effects. Inasmuch as the severity of the stenosis is an important component of the thrombogenic stimulus, comparable and uniform degrees of constriction between treatment groups are required, preferably those in which basal CBF is reduced between 10 and 25% and RH is abolished, or nearly so. It is important to apply these criteria when one is investigating a drug that possesses vasodilatory effects or one whose pharmacologic profile is not completely known. Without exhaustion of the vasodilatory reserve (as evidenced by abolition of RH), elimination of CFRs could result (at least partly) from coronary vasodilation. One difficulty in using RH or basal flow reduction immediately after placing a constrictor on the coronary artery is that CBF starts to decline quickly as platelets accumulate at the site of stenosis and intimal injury. Thus, one needs to assess the degree of flow reduction immediately after constricting the artery. Delaying this assessment will result in an overestimation of the stenosis severity due to accumulation of platelets on the vessel lining. Alternatively, the degree of stenosis can be ascertained by applying the constrictor before denuding the artery (see below), in which case the constrictor (or constrictors) needs to be removed and reapplied. After damaging and stenosing the coronary artery sufficiently, CBF starts declining immediately, reaching zero within 4–12 min, and remaining there until blood flow is restored by manually

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shaking loose the thrombus (“SL,” see Fig. 2.1, bottom). This is usually accomplished by either flicking the Lexan constrictor or sliding the constrictor up and down the artery to mechanically dislodge the thrombus. Spontaneous flow restorations occur under three circumstances: (1) non-severe conditions (i.e., minimal stenosis or de-endothelialization); (2) waning CFRs (which can occur as late as 30–45 mm after establishing CFRs); and/or (3) administration of a partially effective antithrombotic agent. Although the influence of blood pressure on the rate of formation of occlusive thrombi or their stability has not been studied systematically, one might predict that higher arterial pressures would increase the deceleration of CBF to zero by enhancing platelet aggregation through increased shear forces and increased delivery of platelets to the growing thrombus. Higher arterial pressure also might increase the propensity for spontaneous flow restorations before an occlusive thrombus is formed, due to greater stress on the nascent, unconsolidated thrombus. Several groups have examined histologically the coronary arteries harvested from dogs undergoing CFRs usually when CBF is declining or has ceased. Extensive intimal injury, including deendothelialization with adherent platelets and/or microthrombi, is consistently observed. Arteries harvested when CBF is zero invariably reveal a platelet-rich thrombus filling the stenotic segment (19, 21, 23, 28). These histological observations, coupled with the pattern of gradual, progressive declines in CBF and abrupt increases thereof (whether spontaneous or deliberate), provide further evidence that CFRs indeed are caused primarily by platelet thrombi, not vasoconstriction. Although the primary cause of CFRs is platelet aggregation, it is possible that local vasospasm and/or vasoconstriction downstream from the site of thrombosis are induced by vasoactive mediators released by activated and/or aggregating platelets. Experimental evidence supporting vasoconstriction just downstream from the stenosis during CFRs has been demonstrated (29). Further evidence for platelet-dependent thrombus formation in the etiology of CFRs is derived from the pharmacological profile of this model. In general, platelet-inhibitory agents consistently abolish or attenuate CFRs, whereas vasodilators (e.g., nitroglycerin, calcium entry blockers, and papaverine) affect them negligibly (30). Aspirin was the first described inhibitor of CFRs (21). However, in subsequent studies, its effects on CFRs were found to be variable and dose-dependent (22). Variability in the response to aspirin may be related to the severity of the stenosis, as further tightening of the constrictor after an effective dose of aspirin or ibuprofen usually restores CFRs. Prostacyclin, a powerful anti-aggregatory and potent coronary vasodilatory product of endothelial arachidonic acid

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metabolism, is extremely efficacious and potent in abolishing CFRs. It is noteworthy that different drug classes can be compared, as evidenced by the wide range of percentages of responders (28). Advances in platelet physiology and pharmacology have identified a new class of antiplatelet agents that block the platelet membrane glycoprotein IIb/IIIa (GPIIb/IIIa) receptor and hence fibrinogen binding. Fibrinogen binding between platelets is an obligate event in aggregation and is initiated by blood-borne platelet agonists such as ADP, serotonin, thrombin, epinephrine, and collagen (31). The tripeptide sequence Arg–Gly–Asp (RGD), which occurs twice in the Aα-chain of fibrinogen, is believed to mediate, at least in part, the binding of fibrinogen to the GPIIb/IIIa complex. Early experimental results with GPIIb/IIIa antagonists in studies by Coller et al. (32), Bush (26), and Shebuski (25, 33) demonstrated that fibrinogen receptor antagonists are as effective as prostacyclin as anti-aggregatory and antithrombotic agents and do not possess the hemodynamic liabilities associated with prostaglandin-based compounds. Monoclonal antibodies directed against the platelet fibrinogen receptor (abciximab) are essentially irreversible, whereas RGD (tirofiban)- or KGD (eptifibatide)based fibrinogen receptor antagonists are reversible, their effects dissipating within hours after discontinuation of intravenous infusion. The prominence of platelet aggregation vis-à-vis coagulation mechanisms in the Folts model is evidenced by the lack of effect of heparin and thrombin inhibitors reported by most investigators (19, 22). However, heparin and MCI-9038, a thrombin inhibitor, were reported to abolish CFRs in about two-thirds of dogs with recently (30 min) established CFRs, but not in those extent after 3 h (34). The explanation for the differential effects of heparin is not immediately apparent. It may be related to the severity of the stenosis used. These apparently discrepant observations could be related to inhibition of thrombin-stimulated platelet activation and/or aggregation. An attractive feature of the Folts model is its amenability to dose-response studies. Unlike other models in which the thrombotic processes are dynamic, occurring over several minutes to hours, CFRs in the Folts model are repetitive and remarkably unchanging. In the many dogs that received either no intervention or vehicle 1 h after initiating CFRs, flow patterns remained unchanged for at least another hour (23). Thus, one can evaluate several doses of an investigational drug in a single dog. We and others have exploited this to determine potencies, an important basis of comparison between drugs with similar mechanisms of action, thus underscoring another feature of the model: its amenability to quantification of drug response. Two methods for quantifying drug effects in this model have been described.

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Aiken et al. (21) first described a four-point scoring scheme to assess and compare different doses or drugs, ranging from 0 (no effect on CFRs) to 3 (fully effective; complete abolition of CFRs). Intermediate scores of 1 and 2 are respectively applied when the CFR frequency was slowed (but occlusive thrombi still occurred) and when non-occlusive, spontaneously embolizing thrombi were observed. An advantage of this system is the provision of a single number for each evaluation period. A disadvantage is that agents that decrease systemic blood pressure (e.g., prostacyclin) will also decrease coronary perfusion pressure; the coronary flow pattern will be affected, making the scoring system somewhat more subjective. Another method of quantifying CFRs, described by Bush et al. (23), addresses the frequency, expressed on a per hour basis, and severity, based on the average nadir of CBF before a flow restoration. This system is less subjective, but it produces two values per evaluation period, and combinations of the two in an effort to provide a single parameter are awkward. In practical terms, both methods for quantifying CFRs described above provide similar answers. The important point for both is consistency in scoring. This end is best served by well-defined and communicated criteria. To date, the Folts model has been used only to evaluate antithrombotic drugs. No description of this model for the evaluation of thrombolytic drugs or adjunctive agents has been made. However, preliminary data reveal these thrombi to be resistant to doses of thrombolytic agents that lyse thrombi in other models (35). Of all the models described in this review, the thrombi in this model are probably the most platelet-rich and possess relatively less fibrin than, for example, the copper coil or wire models. However, it may be erroneous to conclude that these thrombi are devoid of fibrin, as the fibrinogen that links platelets during aggregation via the GPIIb/IIIa receptor is theoretically capable of undergoing fibrin formation. Several investigators have shown that the same combination of severe vessel narrowing and de-endothelialization results in CFRs in arteries other than the coronary. We have elicited CFRs in femoral arteries in anesthetized dogs with similar degrees of vessel narrowing and deliberate denudation of the artery (unpublished observation). Folts et al. (24) demonstrated that CFRs can be produced in conscious dogs with chronically implanted R Lexan coronary constrictors and flow probes. CFRs were prevented in the interim between implantation and acute study by the administration of aspirin. Al-Wathiqui (36) and Gallagher and co-workers (37) have demonstrated that progressive carotid or coronary arterial narrowing with ameroid constrictors will result in CFRs days to weeks after surgical implantation. These dogs apparently did not undergo deliberate vessel denudation at the

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time of implantation. Perhaps focal inflammation developed in the intervening week(s) between the surgery and the development of CFRs in these animals. Alternatively, there was sufficient intimal vessel injury during implantation of the ameroid constrictors to induce development of CFRs at a later time. CFRs have also been elicited in the renal (38) and carotid (39) arteries of cynomolgus monkeys. Eidt et al. (40) showed that conscious dogs equipped with the same constrictors over segments of the LAD showing endothelial injury undergo repetitive CFRs in response to exercise, but not ventricular pacing. The frequency and severity of CFRs varied more in this model, and some CFRs were non-occlusive. CFRs of most dogs eventually deteriorated to persistent no- or lowflow states. Unlike the open-chest preparation, flow restorations observed in this model occurred spontaneously. Also, the severity of the stenosis produced was not as great as that produced by most practitioners of the Folts model, as reflected by the ability of CBF to increase above control levels initially during exercise. In summary, the Folts model of platelet-dependent thrombus formation is a well-established method to determine the pharmacology of antithrombotic agents. It represents an excellent choice for initial evaluation of antiplatelet activity in vivo, regardless of the artery used. Qualitatively, the thrombogenic stimuli in the Folts model and those responsible for unstable angina may be similar, since an involvement by platelets has clearly been demonstrated in the model and is strongly suspected clinically. It should be remembered, however, that flow restorations in the Folts model require vigorous shaking. In contrast, unstable angina is believed not to involve persistent, total thrombotic coronary occlusion. On the basis of the model’s pharmacological profile, the thrombi in this model also do not appear to resemble those usually responsible for acute myocardial infarction, as the former appear to be unresponsive to thrombolytic agents. The preliminary observations that either streptokinase (SK) or tissue plasminogen activator (t-PA) does not lyse thrombi in the Folts model contrast with the 50–75% response rate to thrombolytic therapy in patients with evolving myocardial infarction (35). However, it is tempting to speculate that the platelet-rich thrombi produced in this model are more like thrombi in those patients whose coronary arteries are not reopened by even early intervention and/or high doses of t-PA (41), and thus could represent a model of “thrombolytic-resistant” coronary thrombosis. In order to study new drugs for their antithrombotic potential in coronary arteries, Folts and Rowe (42) developed a model of periodic acute platelet thrombosis and CFRs in stenosed canine coronary arteries. Uchida described a similar model in 1975 (27). The model includes various aspects of unstable angina pectoris, i.e., critical stenosis, vascular damage, downstream vasospasm

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induced by vasoconstrictors released or generated by platelets. The cyclic variations in CBF are a result of acute platelet thrombi which may occlude the vessel but which either embolize spontaneously or can easily be embolized by shaking the constricting plastic cylinder. CFRs are not a result of vasospasm (24). Clinically, aspirin can reduce the morbidity and mortality of coronary thrombotic diseases but its effect is limited. Similarly, CFRs in the Folts model are abolished by aspirin but the effect can be reversed by increases in catecholamines and shear forces (43). As part of an expert meeting on animal models of thrombosis, a review of the Folts model has been published (44). In this section, five different protocols are described for the induction of coronary thrombosis. 2.1.1.1. Coronary Thrombosis Induced by Stenosis (Protocols 1–4)

The first four protocols are characterized by episodic, spontaneous decreases in CBF interrupted by restorations of blood flow. These alterations in CBF, or CFRs, are associated with transient platelet aggregation at the site of the coronary constriction and an abrupt increase in blood flow after embolization of platelet-rich thrombi. Damage to the vessel wall is achieved by placing a hemostatic clamp on the coronary artery. A fixed amount of stenosis is produced by an externally applied obstructive plastic cylinder at the damaged part of the vessel. In dogs, stenosis is critical; the reactive hyperemic response to a 10-second (s) occlusion is abolished (protocol 1). In pigs, stenosis is subcritical; partial reactive hyperemia remains (45). For some animals, particularly young dogs, damage of the vessel wall and stenosis are not sufficient to induce thrombotic cyclic flow variations. In these cases, additional activation of platelets by infusion of epinephrine (protocol 3) is required, leading to the formation of measurable thrombi. In protocol 4, thrombus formation is induced by subcritical stenosis without prior clamping of the artery and infusion of platelet activating factor (PAF), according to the model described by Apprill et al. (46). In addition to these protocols, coronary spasms induced by released platelet components can influence CBF. Therefore, this model includes the main pathological factors of unstable angina pectoris.

2.1.1.2. Coronary Thrombosis Induced by Electrical Stimulation (Protocol 5)

In this protocol, coronary thrombosis is induced by delivery of low amperage electrical current to the intimal surface of the artery, as described by Romson et al. (47). In contrast to the stenosis protocols, an occluding thrombosis is formed gradually without embolism after some hours. As a consequence of the time course, thrombi formed are of mixed type and contain more fibrin than platelet thrombi formed by critical stenosis.

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2.1.2. Procedure 2.1.2.1. Protocol 1: Critical Stenosis

Dogs of either sex weighing 15–40 kg and at least 8 months of age are anesthetized with pentobarbital sodium (bolus of 30–40 mg/kg and then continuous infusion of approximately 0.1 mg/kg/min); respiration is maintained through a tracheal tube using a positive pressure respirator. The heart is exposed through a left thoracotomy at the fourth or fifth intercostal space; the pericard is opened and the left circumflex coronary artery (LCX) is exposed. An electromagnetic or Doppler flow probe is placed on the proximal part of the LCX to measure CBF. Distal to the flow probe, the vessel is squeezed with a 2-mm hemostatic clamp for 5 s. A small cylindrical plastic constrictor 2–4 mm in length and with an internal diameter of 1.2–1.8 mm (depending on the size of the LCX) is then placed around the artery at the site of the damage. Usually, the constrictor has to be changed several times (2–5 times) until the appropriate narrowing of the vessel is achieved and cyclic flow variations are observed. In the event of occlusion of the artery without spontaneous embolization of the formed thrombus, reflow is induced by shortly lifting the vessel with a thread placed beneath the stenotic site. Only dogs with regularly spaced CFRs of similar intensity within a pre-treatment phase of 60 min are used in these experiments. The test substance is administered by i.v. bolus injection or continuous infusion, or by intraduodenal application. CFRs are registered for 2–4×60 min and compared to pre-treatment values. Prior to testing, preparations for additional hemodynamic measurements are performed (see below).

2.1.2.2. Protocol 2: Subcritical Stenosis

Male castrated pigs (German landrace weighing 20–40 kg) are anesthetized with ketamine (2 mg/kg i.m.), metomidate (10 mg/kg i.p.), and xylazine (1–2 mg/kg i.m.). In order to maintain the stage of surgical anesthesia, animals receive a continuous i.v. infusion of 0.1–0.2 mg/kg/min pentobarbital sodium. Respiration is maintained through a tracheal tube using a positive pressure respirator. The heart is exposed through a left thoracotomy at the fourth and fifth intercostal space; the pericard is opened and the LAD is exposed. An electromagnetic or Doppler flow probe is placed on the proximal part of the LAD to measure CBF. Distal to the flow probe, the vessel is squeezed with a 1-mm hemostatic clamp for 5 s. A small cylindrical plastic constrictor 2 mm in length is then placed around the artery at the site of damage. Usually, the constrictor has to be changed several times until the appropriate narrowing of the vessel is achieved that produces CFRs. CFRs are similar to those in dogs; pigs, however, show a reactive hyperemic response. If embolization

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does not occur spontaneously, the formed thrombus is released by reducing blood flow by shortly lifting the vessel with forceps. Only pigs with regularly spaced CFRs of similar intensity within a pre-treatment phase of 60 min are used for the experiments. The test substance is administered by i.v. bolus injection or continuous infusion, or by intraduodenal application. CFRs are registered for 2×60 min and compared to pre-treatment values. 2.1.2.3. Protocol 3: Stenosis Plus Epinephrine Infusion

If protocol 1 does not lead to CFRs, additional epinephrine (0.2 μg/kg/min) is infused into a peripheral vein for 2×60 min (60 min before and 60 min after drug administration). CFRs are recorded, and the 60-min post-drug phase is compared to the 60-min pre-drug phase.

2.1.2.4. Protocol 4: Stenosis Plus PAF Infusion

The LCX is stenosed without prior mechanical wall injury. This preparation does not lead to thrombus formation (subcritical stenosis). For the induction of CFRs, PAF (C16-PAF, Bachem) (0.2 nmol/kg/min) is infused into one cannulated lateral branch of the coronary artery. After 30 min, PAF infusion is terminated and blood flow returns to a normal, continuous course. Thirty minutes later, the test substance is administered concomitantly with the initiation of a second PAF infusion for 30 min. CFRs are recorded and the drug treatment/second PAF phase is compared to the pre-drug/first PAF phase.

2.1.2.5. Protocol 5: Electrical Stimulation

The LCX is punctured distal to the flow probe with a chrome– vanadium–steel electrode (3 mm in length, 1 mm diameter). The electrode (anode) is placed in the vessel in contact with the intimal lining and connected over a teflon-coated wire to a 9-volt (V) battery, a potentiometer, and an amperemeter. A disc electrode (cathode) is secured to a subcutaneous thoracal muscle layer to complete the electrical circuit. The intima is stimulated with 150 μA for 6 hours (h). During this time, an occluding thrombosis is gradually formed. The test substance, or vehicle as a control, is administered either at the start of the electrical stimulation or 30 min after the start. The time until thrombotic occlusion of the vessel occurs and the thrombus size (wet weight measured immediately after removal at the end of the experiment) are determined. Prior to testing, preparations for additional hemodynamic measurements are performed (see below). For all protocols the following preparations and measurements are performed: 1. To measure peripheral arterial blood pressure (BP) [mm Hg], the right femoral artery is cannulated and connected to a Statham pressure transducer.

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2. Left ventricular pressure (LVP) [mm Hg] is determined by inserting a micro tip catheter via the carotid artery retrogradely. 3. Left ventricular end-diastolic pressure (LVEDP) [mm Hg] is evaluated through sensitive amplification of the LVP. 4. Contractility (LV dp/dt max) [mm Hg/s] is determined from the initial slope of the LVP curve. 5. Heart rate [min–1] is determined from the pulsatile blood pressure curve. 6. The ECG is recorded in lead II. 7. Arterial pH and concentrations of blood gases are maintained at physiological levels by adjusting respiration and infusion of sodium bicarbonate. 8. Blood hematocrit values (37–40%) and number of erythrocytes are kept constant by infusion of oxypolygelatine in dogs and electrolyte solution in pigs. 9. Body temperature is monitored with a rectal thermistor probe and kept constant by placing the animals on a heated metal pad with automatic temperature regulation. 10. Template buccal mucosal bleeding time is carried out using the Simplate device. 11. At the end of the test, animals are sacrificed by an overdose of pentobarbital sodium. For detailed applications of the Folts model, see Folts (44), Folts and Rowe (42, 43), and Folts et al. (19, 24). 2.1.3. Evaluation

For all protocols, the mean maximal reduction of blood pressure (systolic/diastolic) [mm Hg] is determined.

2.1.3.1. Protocols 1–4

The following parameters are determined to quantify stenosisinduced coronary thrombosis: 1. Frequency of CFRs = cycle number per unit time 2. Magnitude of CFRs = cycle area [mm2 ] (total area of all CFRs per unit time, measured by planimetry) 3. Percent change in cycle number and cycle area after drug treatment is calculated relative to pre-treatment controls. 4. Statistical significance is assessed by the paired Student’s t-test.

2.1.3.2. Protocol 5

The following parameters are determined to quantify electrically induced coronary thrombosis: 1. Occlusion time [min] = time to zero blood flow. 2. Thrombus size [mg] = wet weight of the thrombus immediately after removal.

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3. Percent change in mean values for occlusion time and thrombus size in drug-treated groups is calculated relative to the control group. 4. Statistical significance is assessed by the non-paired Student’s t-test. 2.1.4. Critical Assessment of the Method

The stenosis (Folts) and electrical (Romson/Lucchesi) models of coronary thrombosis are widely used to study the role of mediators in the thrombotic process and the effect of new antithrombotic drugs. Bush and Patrick (28) provide an excellent review of the role of the endothelium in arterial thrombosis and the use of the Folts model to determine the effects of thrombosis inhibitors and mediators, i.e., thromboxane, prostacyclin, cyclooxygenase, serotonin, nitric oxide (NO) donors, and other vasodilators. The effect of an NO donor could be reversed by the NO scavenger oxyhemoglobin, which indicated that NO indeed mediates antithrombotic drug action (48). These coronary thrombosis models have recently been used to elucidate the mechanisms of action of several antithrombotic drugs, including the oral GPIIb/IIIa antagonist DMP 728 (49); the low molecular weight heparin (LMWH) enoxaparin (50), which, in contrast to unfractionated heparin, inhibited CFRs; the thrombin inhibitor PEG–hirudin (51); melagatran (52), an anti-P-selectin antibody (53); and activated protein C (54). The clinical relevance of the Folts model has been questioned because the model is very sensitive to antithrombotic compounds. However, in this model, lack of a reversal by epinephrine or an increase in degree of stenosis is able to differentiate any new drug from aspirin. Electrical coronary thrombosis is less sensitive (i.e., aspirin has no effect) and higher doses of some drugs are required. However, in principle, most drugs act in both models, if at all.

2.1.5. Modifications of the Method

Romson et al. (55) described a simple technique for the induction of coronary artery thrombosis in the conscious dog by delivery of low amperage electric current to the intimal surface of the artery. Benedict et al. (56) modified the electrical stimulation of thrombosis model by using two Doppler flow probes proximal and distal to the needle electrode in order to measure changes in blood flow velocity. The electrical current was stopped when a 50% increase in flow velocity was reached, at which point thrombosis occurred spontaneously. Using this model, the investigators demonstrated the importance of serotonin by measuring increased coronary sinus serotonin levels just prior to occlusion. Warltier et al. (57) described a canine model of thrombininduced coronary artery thrombosis to analyze the effects of intracoronary SK on regional myocardial blood flow, contractile function, and infarct size. Al-Wathiqui et al. (36) described

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the induction of CFRs in the coronary, carotid, and femoral arteries of conscious chronically instrumented dogs. The Folts thrombosis model has also been applied to carotid arteries in monkeys. Coller et al. (58) induced CFRs in the carotid arteries of anesthetized cynomologus monkeys and demonstrated that they were abolished by the GPIIb/IIIa antibody abciximab. 2.2. Stenosis- and Mechanical Injury-Induced Arterial and Venous Thromboses (Harbauer Model) 2.2.1. Purpose and Rationale

Harbauer (59) first described a venous model of thrombosis induced by mechanical injury and stenosis of the jugular vein. In a modification of the technique, both arterial and venous thromboses are produced in rabbits by stenosis of the carotid artery and the jugular vein with simultaneous mechanical damage of the endothelium. This results in the activation of platelets and the coagulation system and leads to changes in the bloodstream pattern. As a consequence, occluding thrombi are formed and detected by blood flow measurements. The dominant role of platelets in this model is evidenced by the inhibitory effect of an antiplatelet serum in both types of vessels (60). The modified Harbauer model is used to evaluate the antithrombotic activity of compounds in an in vivo model of arterial and venous thromboses in which thrombus formation is highly dependent on platelet activation.

2.2.2. Procedure

Male Chinchilla rabbits weighing 3–4 kg receive test compound or vehicle as a control by oral, i.v., or i.p. administration. The first ligature (vein; for preparation, see below) is performed at the end of the absorption period (i.p., approximately 30 min; p.o., approximately 60 min; i.v., variable). Sixty-five minutes before stenosis, the animals are sedated by R i.m. injection of 8 mg/kg xylazine (Rompun ) and anesthetized by i.v. injection of 30–40 mg/kg pentobarbital sodium 5 min later. During the course of the test, anesthesia is maintained by continuous infusion of pentobarbital sodium (30–40 mg/kg/h) into one femoral vein. A Statham pressure transducer is placed into the right femoral artery for continuous measurement of blood pressure. Spontaneous respiration is maintained through a tracheal tube. One jugular vein and one carotid artery are exposed on opposite sides. Small branches of the vein are clamped to avoid blood flow around the vessel occlusion.

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Electromagnetic or Doppler flow probes are placed on the vein (directly central to the vein branching) and on the artery (as centered as possible). Blood flow [ml/min] is measured continuously. After blood flow reaches a steady state (approximately 15–30 min), a metal rod with a diameter of 1.3 mm is placed on the jugular vein (2 cm central to the vein branching) and a ligature is tightened. After 1 min, the rod is removed from the ligature. Immediately thereafter (approximately 1.5 min), the carotid artery is damaged by briefly squeezing it with forceps. A small plastic constricting cylinder (2 mm in length and 1.2 mm in diameter) is placed around the site of endothelial damage. Template bleeding time is measured at various time intervals before and after drug treatment (depending on the route of R administration) in the shaved inner ear using a Simplate device. Care is taken to select parts of the skin without large vessels. 2.2.3. Evaluation

1. Percent thrombus formation (thrombosis incidence) is determined as the number of occluded vessels (blood flow=0). 2. Percent inhibition of thrombosis is calculated in each dosage group relative to the respective vehicle controls. 3. Thrombosis incidence in the vehicle controls is set as 100%. 4. Statistical significance is assessed by means of the Fisherexact-test. 5. If initial values for blood flow do not significantly differ in the dosage and control groups, the area below the blood flow curve is measured by planimetry, and the mean value of each dosage group is compared to the control using the unpaired Student’s t-test. 6. Mean occlusion time [min] in the dosage and control groups are calculated and compared using the Students’s t-test. 7. The maximal change in systolic and diastolic blood pressure during the time period of stenosis as compared to the initial values before drug administration is determined. There is no standardized assessment score. For example, a reduction of systolic blood pressure by 30 mmHg and diastolic blood pressure by 20 mmHg is generally accepted as a strong reduction in blood pressure.

2.2.4. Critical Assessment of the Method

Two main factors of arterial thrombosis in human are essential components of this model: high-grade stenosis and vessel wall damage. In the absence of either, no thrombus is formed. The occlusive thrombus is formed fast and in a highly reproducible manner. In both vessels, thrombus formation is dependent on platelet function, as shown by the effects of antiplatelet serum.

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Thus, jugular vein thrombosis in this model differs from stasisinduced deep vein thrombosis with prominent fibrin formation. On the other hand, occlusive thrombi are more stable than the pure platelet thrombi in the Folts model (see Section 2.1), as carotid blood flow cannot be restored by shaking the constrictor. The following antithrombotic drugs have been shown to be effective in this model: (1) antiplatelet drugs such as ticlopidine, prostacyclin/iloprost, NO donors (SNP, molsidomine), but not aspirin or thromboxane synthetase inhibitors; (2) anticoagulants such as hirudin, high-dose heparin, and warfarin; and (3) SK/t-PA (60, 61, and unpublished data). Drugs that simply lower blood pressure, such as hydralazine, clonidine, and prazosin have no effect on thrombus formation in this model. 2.2.5. Modifications of the Method

Bevilacqua et al. (61) applied this model to the rabbit carotid arteries and compared one artery before drug treatment with the contralateral artery after drug treatment. Heparin, the synthetic thrombin inhibitor FPRCH2 Cl, iloprost, and t-PA, but not aspirin, inhibited carotid occlusion in this model. Spokas and Wun (62) induced venous thrombosis in the vena cava of rabbits by vascular damage and stasis. Vascular wall damage was achieved by crushing the vessel with hemostat clamps. A segment of the vena cava was looped with two ligatures 2.5-cm apart, and then 2 h after ligation, the isolated venous sac was dissected and the clot was removed for determination of dry weight. Lyle et al. (63), in pursuit of an animal model that mimicked thrombotic re-occlusion and restenosis after successful coronary angioplasty in human, developed a model of angioplasty-induced injury in atherosclerotic rabbit femoral arteries. Acute 111 indiumlabeled platelet deposition and thrombosis were assessed 4 h after balloon injury in arteries subjected to prior endothelial damage (by air desiccation) and cholesterol supplementation (one month). The effects of inhibitors of FXa or platelet adhesion, heparin, and aspirin on platelet deposition were studied. Meng (64), Meng and Seuter (65), and Seuter et al. (66) described a method to induce arterial thrombosis in rats by chilling of the carotid artery (thrombosis induced by super cooling). Rats were anesthetized, and then the left carotid artery was exposed and occluded proximal by means of a small clamp. The artery was placed for 2 min into a metal groove that was cooled to –15◦ C. The vessel was then compressed using a weight of 200 g. In addition, a silver clip was fixed to the vessel distal to the injured area to produce disturbed and slow blood flow. After 4 min, the proximal clamp was removed and blood flow was reestablished in the injured artery. A similar model in the rabbit has also been developed, with slightly different conditions (chilling temperature of –12◦ C for 5 min, and a compression weight of 500 g). The wound is closed and the animal is allowed to recover from

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anesthesia. Antithrombotic compounds are administered in various doses at different time intervals before surgery. After 4 h, the animals receive heparin and are re-anesthetized. The lesioned carotid artery is removed and thrombus wet weight is immediately measured. 2.3. Electrically Induced Thrombosis

A novel technique for inducing arterial thrombosis was introduced by Salazar (67) in which anodal current was delivered to the intravascular lumen of a coronary artery in the dog via a stainless steel electrode. The electrode was positioned under fluoroscopic control, which somewhat complicated the procedure. Subsequently, Romson et al. (55) modified the procedure such that the electrode was placed directly into the coronary artery of an open-chest, anesthetized dog. This technique then allows one to produce a thrombus in the anesthetized animal or to close the chest after inserting the electrode and allow the animal to recover, after which thrombosis can be elicited later in the conscious animal. The advantage of this modification is that it allows induction of thrombus formation without the need for fluoroscopy. The stimulation electrode is constructed from a 25- or 26gauge stainless steel hypodermic needle tip, which is attached to a 30-gauge teflon-insulated silver-coated copper wire. Anodal current is delivered to the electrode via either a 9-V nickel–cadmium battery with the anode connected in series to a 250,000-ohm potentiometer or with a Grass stimulator connected to a Grass constant current unit and a stimulus isolation unit. The cathode in both cases is placed into a subcutaneous site completing the circuit. The anodal current can be adjusted to deliver 50–200 μA. Anodal stimulation results in focal endothelial disruption, which in turn induces platelet adhesion and aggregation at the damaged site. This process is then followed by further platelet aggregation and consolidation, with the growing thrombus entrapping red blood cells. A modification of the method of Romson et al. (55) involves placement onto the coronary artery of an external, adjustable occluder (68) to produce a fixed stenosis on the coronary artery. A flow probe to record CBF is placed on the proximal portion of the artery followed by the stimulation electrode, with the clamp being placed most distally (Fig. 2.2). The degree of stenosis can then be controlled by adjusting the clamp. The resulting stenosis is produced in an effort to mimic the human pathophysiology of atherosclerotic coronary artery disease, whereby thrombolytic therapy restores CBF through a coronary artery with residual narrowing due to atherosclerotic plaque formation. Another modification of the electrical stimulation model that merits discussion is described by Benedict et al. (56). They discontinued anodal current when mean distal coronary flow velocity (measured with Doppler flow meter) increased by approximately

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Fig. 2.2. Model of coronary artery thrombosis in the dog. Electrical injury to the intimal surface of the artery leads to occlusive thrombus formation. The thrombus is formed in the presence of a flow-limiting stenosis induced by a Goldblatt clamp. Upon spontaneous occlusion, heparin is administered and the clot is aged for 1 h before initiating the t-PA infusion.

50%, reflecting disruption of normal axial flow by the growing thrombus. Occlusive thrombosis occurred within 1 h after stopping the current (2 h after starting the current). In these studies, coronary sinus plasma levels of serotonin, an index of intravascular platelet aggregation, were increased approximately 20-fold just before occlusive thrombus formation. The results of these studies agree with others in showing that either proximal flow velocity or electromagnetically measured CBF declines trivially over the majority of the time period in which the thrombus is growing. The largest declines in (volume) flow occur over a small and terminal fraction of the period between initial vessel perturbation and final occlusion. During that interval, coronary lumenal area decreases rapidly and to a critical degree, as platelets accrue at the growing thrombus. The studies by Benedict et al. (56) demonstrate that this final phase of thrombosis can occur independently of electrical stimulation. This variation of the model may be attractive to those who wish to produce occlusive thrombosis without continued electrical stimulation.

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Regardless of whether electrical stimulation is continued until occlusive thrombosis, there is another component to this model that has upside and downside potential, namely, the opportunity for coronary vasoconstriction to occur. Although the incidence of Prinzmetal’s angina is low, it is widely suspected that vasospasm superimposes on a primarily thrombotic event in unstable angina and myocardial infarction. In studies by Van der Giessen et al. (69), nifedipine was reported to increase the extent of CBF after plasmin-induced thrombolysis in a porcine model of electrically induced coronary thrombosis. In their model, the anodal stimulation was applied circumferentially to the exterior surface of the LAD, and an external constrictor was not used. Depending on the hypothesis being tested, the experimenter can leave intact or minimize this potential through the use or disuse of an external constrictor. As in the Folts and Gold coronary thrombo(ly)sis models, blood pressure must be taken into account or maintained within acceptable limits, since, in the presence of a critical stenosis, auto-regulation no longer exists. Under these conditions CBF is highly dependent on driving pressure (arterial pressure). Numerous experimental studies evaluating anticoagulants, antithrombotic, and/or thrombolytic drugs have been performed using this model. In the initial report by Romson et al. (55), the cyclooxygenase inhibitor ibuprofen was evaluated. Comparison of myocardial infarct size, thrombus weight, arrhythmia development, and scanning electron microscopy of drug-treated and control animals indicated that ibuprofen protected the conscious dog against the deleterious effects of coronary artery thrombosis. Subsequent studies in the same model and laboratory evaluated the antithrombotic potential of various TXA2 synthetase inhibitors, such as U 63557A, CGS 13080, OKY 1581, and dazoxiben. When the TXA2 synthetase inhibitors were administered before induction of the current, OKY 1581 (70) and CGS 13080 (71) reduced the incidence of coronary thrombosis, whereas U 63557A (72) and dazoxiben (73) were ineffective and partially effective, respectively. The differences in efficacy noted among the TXA2 synthetase inhibitors were ascribed to differences in potency and duration of action. Other investigators have used this model to study the prevention of original coronary thrombosis in the dog. Fitzgerald et al. (74) studied the TXA2 synthetase inhibitor U 63557A alone or in combination with L-636,499, an endoperoxide/thromboxane receptor antagonist. U 63557A alone did not prevent coronary thrombosis when administered before current application, whereas the combination of U 63557A and L-636,499 was highly effective. These data suggest that prostaglandin endoperoxides may modulate the effects of TXA2 synthetase inhibitors and that this response may be blocked by concurrent administration of

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an endoperoxide/thromboxane receptor antagonist. The murine monoclonal antibody to platelet GPIIb/IIIa (7E3) was studied in this model for its ability to prevent thrombus formation. At a dose of 0.8 mg/kg i.v., the 7E3 monoclonal antibody completely prevented original thrombus formation (75). In addition to the evaluation of antithrombotic (i.e., antiplatelet) agents, the electrical injury model is useful for studying anticoagulant FXa inhibitors, such as YM 60628 (86), and thrombolytic drugs. When evaluating thrombolytic agents, the thrombus is allowed to form without drug intervention and then aged for various periods. Schumacher et al. (68) demonstrated that intracoronary SK was an effective thrombolytic drug in this model; the thrombolytic effectiveness being augmented by the concurrent administration of heparin and prostacyclin, or by a TXA2 synthetase inhibitor (68). In other studies reported by Shebuski et al. (76), the TXA2 receptor antagonist BM 13.177 hastened t-PA-induced thrombolysis and prevented acute thrombotic re-occlusion. Van der Giessen et al. (77) subsequently demonstrated that BM 13.177 prevented original thrombus formation in 75% of pigs undergoing electrical stimulation; aspirin was ineffective in this porcine model. These and other studies underscore the potential for adjunctive therapy to hasten thrombolysis and/or prevent re-occlusion, both contributing to greater salvage of ischemic myocardium. Like the copper coil model, the electrical stimulation model has been used to produce experimental myocardial infarction. Patterson et al. (78) have used this technique to produce coronary thrombosis in the LCX (which supplies blood flow to the posterior LV wall in dogs) in dogs with a previous anterior wall infarct to mimic sudden cardiac death that occurs in people during a second (recurrent) myocardial infarction or ischemic event. This model has also been modified to demonstrate the efficacy of adjuncts to thrombolytic therapy (79–82). In this case, the thrombus is allowed to extend until it completely occludes the vessel. Usually, the thrombus is allowed to stabilize, or “age,” to mimic the clinical setting in which a time lag exists between the thrombotic event and the pharmacological intervention. At the end of the stabilization period, thrombolytic agents such as t-PA or SK are administered in conjunction with the novel antithrombotic agent to lyse the thrombus and maintain vessel patency. The incidence and times of reperfusion and re-occlusion are the major endpoints. These studies have established that recombinant tick anticoagulant peptide (rTAP), a potent and selective FXa inhibitor derived from the soft tick (83), promotes rapid and prolonged reperfusion at doses that produce relatively minor elevations in PT, aPTT, and template bleeding time.

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2.3.1. Purpose and Rationale

The use of an electrical current to induce thrombosis in hamster and dog was described in the early 1950s by Lutz et al. (84) and Sawyer et al. (85, 86). In general, two different approaches are taken in this model. One method produces electrical damage by means of two externally applied hook-like electrodes (87, 88). The other method uses a needle electrode that is advanced through the walls of the blood vessel and positioned in the lumen; a second electrode is placed at a subcutaneous site to complete the circuit (55, 56, 67).

2.3.2. Procedure

Anaesthetized rats weighing 200–300 g are intubated and a femoral artery is cannulated for administration of test compound(s). One carotid artery is isolated from the surrounding tissue over a distance of 10–15 mm. A pair of rigid stainless steel wire hook-like electrodes with a working distance of 4 mm is positioned on the artery by means of a rack and pinion gear manipulator. The artery is raised slightly away from the surrounding tissue. Isolation of the electrodes is achieved by the insertion of a small piece of parafilm under the artery. Blood flow is measured with an ultrasonic Doppler flow meter (Transonic, Ithaca NY, USA); the flow probe (1RB) is placed proximal to the damaged area. Thrombus formation is induced in the carotid arteries by the application of an electrical current (350 V, DC, 2 mA) delivered by an electrical stimulator (Stoelting Co, Chicago, Cat. No 58040) for 5 min to the exterior surface of the artery.

2.3.3. Evaluation

1. Blood flow before and after induction of thrombus for 60 min. 2. Time to occlusion [min] = the time between onset of the electrical current and the time at which blood flow decreases to less than 0.3 ml/min. 3. Patency of the blood vessel over 30 min.

2.3.4. Critical Assessment of the Method

Thrombi formed by electrical induction are composed of densely packed platelets, with some red cells. Moreover, electrical injury causes extensive damage to intimal and sub-intimal layers. The endothelium is completely destroyed, and the damage extends to sub-endothelial structures, including smooth muscle cells. This deep damage could reduce sensitivity in terms of discriminating between drugs on the basis of their antithrombotic activity. However, Philp et al. (88) showed that unfractionated heparin completely blocks thrombus formation, whereas other antiplatelet agents exhibit differential antithrombotic actions. The investigators concluded that this relatively simple model of arterial thrombosis might prove to be a useful screening test for drugs with antithrombotic potential.

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2.3.5. Modifications of the Method

In a modification of this model by Salazar (67), a stainless steel electrode is inserted into a coronary artery in the dog to deliver anodal current to the intravascular lumen. The electrode is positioned under fluoroscopic control, complicating the procedure somewhat. Romson et al. (55) described a further modification in which the electrode was placed directly into the coronary artery of open-chest anaesthetized dogs. Rote et al. (89, 90) applied the carotid thrombosis model to dogs. A calibrated electromagnetic flow meter was placed on each common carotid artery proximal to the point of insertion of an intravascular electrode and a mechanical constrictor. The external constrictor was adjusted with a screw until the pulsatile flow pattern was decreased by 25% without alteration in mean blood flow. Electrolytic injury to the intimal surface was accomplished with an intravascular electrode composed of a teflon-insulated silvercoated copper wire connected to the positive pole of a 9-V nickel– cadmium battery in series with a 250,000-ohm variable resistor. The cathode was connected to a subcutaneous site. Injury was initiated in the right carotid artery by application of a 150-μA continuous pulse anodal direct current to the intimal surface of the vessel for a maximum duration of 3 h, or for 30 min beyond the time of complete vessel occlusion, as determined by blood flow recordings. Upon completion of the study on the right carotid, the procedure was repeated on the left carotid artery after administration of test drug. Benedict et al. (56) introduced a procedure in which anodal current was discontinued when mean distal coronary flow velocity increased by approximately 50%, reflecting disruption of normal flow by the growing thrombus. An occlusive thrombosis occurred within 1 h after cessation of the electrical current. In this model, the final phase of thrombosis occurred independently of electrical injury. A ferret model of acute arterial thrombosis was developed by Schumacher et al. (91). A 10-min anodal electrical current of 1 mA was delivered to the external surface of the carotid artery while measuring carotid blood flow. This produced an occlusive thrombus in all vehicle treated ferrets within 41±3 min with an average weight of 8±1 mg. Thrombus weight was reduced by aspirin or a thromboxane receptor antagonist. Guarini (92) reported the formation of a completely occlusive thrombus in the common carotid artery of rats by applying an electrical current to the arterial wall (2 mA for 5 min) while simultaneously constricting the artery with a hemostatic clamp placed immediately downstream from the electrodes.

2.4. Ferric Chloride (FeCl3 )-Induced Thrombosis

The administration of a variety of chemicals either systemically or locally can result in damage to the endothelium with

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subsequent generation of a thrombus. Such compounds include ferric/ferrous chloride, fluorescein-labeled dextran and Rose Bengal. In models employing ferric (ferrous) chloride (93), the carotid artery of rats is isolated. A flow probe is placed proximal to the intended site of lesion and a 3-mm disc of filter paper which has been soaked in ferric/ferrous chloride (35-50%) is placed on the artery. The application of ferric (ferrous) chloride results in transmural vascular injury leading to the formation of occlusive thrombi. This injury is believed to be a result of lipid peroxidation catalyzed by the ferric (ferrous) chloride. Thrombus formation, measured as a decrease in blood flow through the vessel, typically occurs within 30 min. Microscopic analysis of the thrombi has shown them to be predominantly platelet-rich clots. This model has been used to study the antithrombotic effects of direct thrombin inhibitors (94–96) and heparins. Endothelial damage can also be induced by fluoresceinor fluorescein isothiocyanate (FITC)-conjugated compounds. A model has been described in which FITC–dextran is administered intravenously to mice. Thrombus formation is induced upon exposure of the microvessels of the ear to the light of a mercury lamp (excitation wavelength of 450–490 nm) (97). The endothelial damage induced in this model is believed to be a result of the generation of singlet molecular oxygen produced by energy transfer from the excited dye (98). Thrombus formation is measured using intravital fluorescence microscopy. This detection technique allows for a number of endpoints to be quantitated, including changes in luminal diameter due to thrombus formation, blood flow measurements, and extravasation of the FITC–dextran. This model offers the advantages of not requiring surgical manipulations, which can cause hemodynamic or inflammatory changes, allowing for repeated analysis of the same vessel segments over time, and being applicable to the study of both arteriolar and venular thromboses. The administration of Rose Bengal has been used similarly (99). 2.4.1. Purpose and Rationale

A variety of chemical agents have been used to induce thrombosis in animals. The use of topical FeCl3 as a thrombogenic stimulus in veins was described by Reimann-Hunziger (100). Kurz et al. (93) demonstrated that the thrombus produced with this method in the carotid arteries of rats is composed of platelets and red blood cells enmeshed in a fibrin network. This simple and reproducible test has been used for the evaluation of antithrombotic (101) and pro-fibrinolytic test compounds (102).

2.4.2. Procedure

Rats weighing 250–300 g are anaesthetized with Inactin (100 mg/kg) and a polyethylene catheter (PE-205) is inserted

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into the trachea via a tracheotomy to facilitate breathing. Catheters are also placed in the femoral artery for blood sampling and measurement of arterial blood pressure and in the jugular vein for administration of test compounds. The right carotid artery is isolated and an ultrasonic Doppler flowprobe (probe 1RB, Transonic, Ithaca NY, USA) is placed on the vessel to measure blood flow. A small piece of parafilm “M” (American Can Co., Greenwich, CT, USA) is placed under the vessel to isolate it from surrounding tissue throughout the experiment. The test compound is administered by gavage or as an i.v. injection at a defined time prior to initiation of thrombus formation. Thrombus formation is induced by the application of a piece of filter paper (2 mm×5 mm) saturated with 25% FeCl3 to the carotid artery. The paper is allowed to remain on the vessel for 10 min and is then removed. Parameters (see below) are monitored for 60 min after the induction of thrombosis, after which the thrombus is removed and weighed. 2.4.3. Evaluation

1. Blood flow before and after induction of thrombus for 60 min 2. Time to occlusion [min]: the time between FeCl3 application and the point at which blood flow decreases to less than 0.3 ml/min 3. Thrombus weight after blotting the thrombus on filter paper

2.5. Thrombin-Induced Clot Formation in Rabbit Femoral or Canine Coronary Artery

Localized thrombosis can also be produced in rabbit peripheral blood vessels such as the femoral artery by injection of thrombin, calcium chloride and fresh blood via a side branch (103). Either femoral artery is isolated distal to the inguinal ligament and traumatized distally from the lateral circumflex artery by rubbing the artery with the jaws of forceps. An electromagnetic flow probe is placed distal to the lateral circumflex artery to monitor femoral artery blood flow (Fig. 2.3). The superficial epigastric artery is cannulated for induction of the thrombus and subsequent infusion of thrombolytic agents. Localized thrombi distal to the lateral circumflex artery with snares approximately 1-cm apart are induced by the sequential injection of thrombin, CaCl2 (1.25 mmol), and a volume of blood sufficient to distend the artery. After 30 min, the snares are released and femoral artery blood flow is monitored for 30 mm to confirm total obstruction of flow by the thrombus. These models are not appropriate for evaluating drugs for their ability to inhibit original thrombosis. However, the model is particularly appropriate for evaluating thrombolytic agents and adjunctive therapies for their ability to hasten and/or enhance lysis or prevent acute re-occlusion after discontinuing administration of a thrombolytic agent.

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Fig. 2.3. Rabbit model of femoral arterial thrombosis. A clot is introduced into an isolated segment of femoral artery by injection of thrombin, CaCl2 , and whole blood. After aging for 1 h, t-PA is infused. Reperfusion is assessed by restoration of blood flow.

2.5.1. Purpose and Rationale

A canine model of thrombin-induced clot formation was developed by Gold et al. (104) in which localized coronary thrombosis was produced in the LAD. This is a variation of the technique described by Collen et al. (105) who used radioactive fibrinogen to monitor the occurrence and extent of thrombolysis of rabbit jugular vein clots. The vessel was intentionally de-endothelialized by external compression with blunt forceps. Snare occluders were then placed proximal and distal to the damaged site, and thrombin (10 U) was injected into the isolated LAD segment in a small volume via a previously isolated side branch. Autologous blood (0.3–0.4 ml) mixed with calcium chloride (0.05 M) was also injected into the isolated LAD segment, producing a stasis-type red clot superimposed on an injured blood vessel. The snares were released 2–5 min later and total occlusion was confirmed by selective coronary angiography. This model of coronary artery thrombosis relies on the conversion of fibrinogen to fibrin by thrombin. The fibrin-rich thrombus contains platelets, but at no greater concentration than in a similar volume of whole blood. Once the thrombus is formed, it is allowed to age for 1–2 h, after which a thrombolytic agent can be administered to lyse the thrombus and restore blood flow.

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2.5.2. Procedure

In the initial study described by Gold et al. (104), recombinant t-PA was characterized for its ability to lyse 2-h-old thrombi. Tissue plasminogen activator was infused at doses of 4.3, 10, and 25 μg/kg/min i.v. and resulted in reperfusion times of 40, 31, and 13 min, respectively. Thus, in this model of canine coronary thrombosis, t-PA exhibited dose-dependent coronary thrombolysis. It is also possible to study the effects of different doses of t-PA on parameters of systemic fibrinolytic activation, such as fibrinogen, plasminogen, and a2-antiplasmin, as well as to assess myocardial infarct size. For example, Kopia et al. (106) demonstrated that SK elicited dose-dependent thrombolysis in this model. Subsequently, Gold et al. (107, 108) modified the model to study not only reperfusion, but also acute re-occlusion. Clinically, re-occlusion is a persistent problem after effective coronary thrombolysis, which is reported to occur in 15–45% of patients (109). Thus, an animal model of coronary reperfusion and re-occlusion would be important from the standpoint of evaluating adjunctive therapies to t-PA to hasten and/or increase the response rate to thrombolysis as well as prevent acute re-occlusion. The model of thrombin-induced clot formation in the canine coronary artery was modified such that a controlled high-grade stenosis was produced with an external constrictor. Blood flow was monitored with an electromagnetic flow probe. In this model of clot formation with superimposed stenosis, reperfusion in response to t-PA occurs with subsequent re-occlusion. The monoclonal antibody against the human GPIIb/IIIa receptor developed by Coller et al. (110) and tested in combination with t-PA in the canine thrombosis model hastened t-PA-induced thrombolysis and prevented acute re-occlusion (111). These actions in vivo were accompanied by abolition of ADP-induced platelet aggregation and markedly prolonged bleeding time.

2.6. Laser-Induced Thrombosis

The physiologic responses to injury in the arterial and venous systems vary in part due to differences in blood flow conditions, leading to different clot compositions. This model of arterial thrombosis is based on the development of a platelet-rich thrombus following laser-mediated thermal injury to the vascular wall. This model was first described by Weichert and Breddin (112). In this model, an intestinal loop of an anesthetized rat is exposed through a hypogastric incision and spread on a microscope stage while being continuously irrigated with sterile physiologic saline. Vascular lesions are induced on small mesenteric arterioles with an argon laser beam (50 mW at microscope, 150-ms duration) directed through the optical path of the microscope. Exposure of the laser beam is controlled by means of a camera shutter. Laser shots are made every minute. Antithrombotic potency is evaluated

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in real time by microscopic evaluation of vascular occlusion. The number of laser injuries required to induce a thrombus with a length of at least 1.5 times the inner diameter of the vessel is taken as an endpoint. The antithrombotic activity of several thrombin inhibitors has been compared to unfractionated heparin using the laser-induced thrombosis model. Each inhibitor was administered intravenously via one of the tail veins and allowed to circulate for 5 min prior to the initiation of the laser-induced lesions. Saline-treated control rats required an average of three laser shots to reach an endpoint. Each thrombin inhibitor produced a dose-dependent antithrombotic effect in this model. In comparing the dose of each agent required to extend the endpoint to six laser shots, heparin was observed to be the most potent antithrombotic agent (0.08 μmol/kg), followed by Ac-(D)-Phe-Pro-boroArgOH (0.154 μmol/kg) and then hirudin (0.28 μmol/kg). Consistent with the results obtained with these agents in the rabbit jugular vein stasis thrombosis model, D-Me-Phe-Pro-Arg-H exhibited the weakest effects in the laser-induced thrombosis model (2 μmol/kg). 2.6.1. Purpose and Rationale

In this model, thrombus formation in rat or rabbit mesenteric arterioles or venules is induced by laser-mediated thermal injury to the vascular wall. The procedure can be performed in normal or pretreated (i.e., induced arteriosclerosis or adjuvant arthritis) animals. In this model, thrombus formation is mediated by a dual mechanism of platelet adhesion to the injured endothelial vessel wall and ADP-induced platelet aggregation. Most likely, ADP is released by erythrocytes that are lysed by the laser, based on the observation that erythrocyte hemoglobin strongly absorbs the frequencies of light emitted by the laser beam. A secondary aggregation stimulus following the release of ADP is mediated by the platelets themselves.

2.6.2. Procedure

2.6.2.1. Equipment

1. 4 W argon laser (Spectra Physics, Darmstadt, FRG) with a wave length of 514.5 nm; energy below the objective of 15 mW; duration of exposure, 1/30 or 1/15 s. 2. Microscope ICM 405, LD-Epipland 40/0.60 (Zeiss, Oberkochen, FRG) 3. Video camera (Sony, Trinicon tube) 4. Recorder (Sony, U-matic 3/4 ) 5. Videoanalyzer to determine blood flow velocity

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2.6.2.2. Experimental Course

2.6.2.3. Standard Compounds

2.6.3. Evaluation

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Male Sprague-Dawley, spontaneously hypertensive stroke-prone Wistar or Lewis rats with adjuvant-induced arthritis weighing 150–300 g are used. Alternatively, New Zealand rabbits with arteriosclerosis induced by cholesterol feeding for 3 months are used. Animals receive test compound by oral, i.v., i.p. or s.c. administration. Control animals are treated with vehicle alone. Prior to thrombus induction, the animals are pretreated by s.c. injection of 0.1 mg/kg atropine sulfate solution and anaesthetized by i.p. administration of 100 mg/kg ketamine hydrochloride and 4 mg/kg xylazine. Thrombus formation is induced 15, 30, 60, or 90 min postdosing. The procedure is carried out in arterioles or venules 13 ± 1 μm in diameter of the fat-free ileocaecal portion of the mesentery. During the procedure, the mesenterium is superfused with a physiological saline solution or degassed paraffin liquid (37◦ C). The ray of the argon laser is guided into the inverted ray path of the microscope by means of a ray adaptation and adjusting device. The frequency of injury is 1 per 2 min. The exposure time for a single laser shot is 1/30 or 1/15 s. The number of injuries necessary to induce a defined thrombus is recorded. All thrombi formed during the observation period with a minimum length of 13 μm or an area of at least 25 μm2 are evaluated. The procedure is photographed using a video system. • acetylsalicylic acid (10 mg/kg, per os) • pentoxifylline (10 mg/kg, per os) For a detailed description and evaluation of various agents and mechanisms, please refer to the following references: Arfors et al. (113); Herrmann (114); Seiffge and Kremer (115, 116); Seiffge and Weithmann (117); and Weichert (118). The number of laser shots required to produce a defined thrombus is determined. Mean values and SEM are calculated. Results are typically presented in graph form.

2.7. Photochemical Induced Thrombosis 2.7.1. Purpose and Rationale

In 1977, Rosenblum and Sabban (119) reported that ultraviolet light can produce platelet aggregation in cerebral microvessels of the mouse after intravascular administration of sodium fluorescein, and demonstrated that in contrast to heparin, aspirin and indomethacin prolonged the time to first platelet aggregation. A detailed study by Herrmann (114) demonstrated that scavengers of singlet oxygen, but not hydroxyl radicals, inhibited platelet

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aggregation induced by photochemical reaction. The investigators postulated that excitation of intravascular fluorescein results in the production of singlet oxygen, which damages endothelial cells and leads to platelet adhesion and aggregation. 2.7.2. Procedure

Studies are performed in mesenteric arteries 15–30 μm in diameter in anesthetized rats. After i.v. injection of 0.3 ml of fluorescein isothiocyanate–dextran 70 (FITC–dextran; 10%) (Sigma), arterioles are exposed to ultraviolet light (excitation, 490 nm; emission, 510 nm).

2.7.3. Evaluation

Thrombus formation is quantitated by determining the time between onset of excitation and appearance of the first platelet aggregate adhering to the vessel wall.

2.7.4. Critical Assessment of the Method

In contrast to other thrombosis induction methods, photochemically induced thrombosis is amenable to use in small animals. Thrombi are composed primarily of platelets, however, the primary target of the photochemical insult is endothelial cells through induced oxygen radical damage.

2.7.5. Modifications of the Method

Matsuno et al. (120) reported a method to induce thrombosis in the rat femoral artery by means of a photochemical reaction after injection of a fluorescent dye (Rose Bengal, 10 mg/kg i.v.) followed by transillumination with a filtered xenon lamp (wave length, 540 nm). Blood flow was monitored by a pulsed Doppler flow meter. Occlusion was achieved after approximately 5–6 min. Pretreatment with heparin prolonged the time required to interrupt the blood flow in a dose-dependent manner. This model has also been used to study the thrombolytic mechanisms of t-PA. For a comparative analysis of hirudin in various models, see Just et al. (45).

2.8. Foreign Surface-Induced Thrombosis

The presence of foreign materials in the circulation results in activation of the coagulation and platelet systems. A variety of prothrombotic surfaces have been used for the development of experimental animal thrombosis models. In contrast to many other thrombosis models, thrombosis induced by foreign surfaces does not presuppose endothelial damage.

2.8.1. Wire Coil-Induced Thrombosis 2.8.1.1. Purpose and Rationale

This classical method of producing thrombosis is based on the insertion of wire coils into the lumen of blood vessels. The model was first described by Stone and Lord (121) using the dog aorta and was further modified for use in arterial coronary vessels of opened-chest dogs. The formation of thrombotic material around

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the coil is reproducible and can be easily standardized for pharmacological studies (48, 122, 123). The use of this model in venous vessels was described by Kumada et al. (124). Venous thrombosis is produced in rats by insertion of a stainless steel wire coil into the inferior caval vein. Platelets and plasmatic coagulation are activated on the wire coil. Thrombus formation on the wire is quantitated by measuring the protein content of the isolated thrombotic material. The kinetics of thrombus formation show an increase in weight and protein content within the first 30 min of insertion, followed by a period of steady state flux between thrombus formation and endogenous thrombolysis, leading to a level protein content of thrombi starting at 1 h and lasting up to 48 h after implantation. The incidence of thrombosis in untreated control animals in this model is 100%. The model is used to evaluate antithrombotic and thrombolytic properties of test compounds in an in vivo model of venous thrombosis in rats. 2.8.1.2. Procedure

2.8.1.3. Evaluation

Male Sprague-Dawley rats weighing 260–300 g receive test compound, or vehicle as a control, by oral, i.v. or i.p. administration. At the end of absorption (i.v., 1 min; i.p., 30 min; p.o., 60 min), the animals are anesthetized by i.p. injection of 1.3 g/kg of urethane. Through a midline incision the caudal caval vein is exposed R and a stainless steel wire coil (Zipperer , size 40; Zdarsky Erler KG, München) is inserted into the lumen of the vein just below the left renal vein branching by gently twisting the wire toward the iliac vein. The handle of the carrier is cut off so as to hold the back end of the wire at the vein wall. The incision is sutured and the animal is placed on its back on a heating pad (37◦ C). The wound is reopened after 2 h and the wire coil with the thrombus on it is carefully removed and rinsed with a 0.9% saline solution. The thrombotic material is dissolved in 2 ml of alkaline sodium carbonate solution (2% Na2 CO3 in 0.1 N NaOH) in a boiling water bath for 3 min. The protein content is determined in 100 μl aliquots by the colorimetric method of Lowry (Fig. 2.4). Thrombolysis. In addition to the procedure described above, a thrombolytic test solution is continuously infused through a polyethylene catheter inserted into the jugular vein. Ninety minutes after wire implantation, the test compound or the vehicle (control) is infused for up to 2.5 h. The wire coil is then removed and the protein content of the thrombus is determined. Using this model, Bernat et al. (125) demonstrated the fibrinolytic activity of urokinase and SK–human plasminogen complex. 1. Thrombosis incidence = number of animals in each dosage group that develop thrombi as compared to the vehicle control.

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Fig. 2.4. Schematic diagram of the canine femoral artery copper coil model of thrombolysis. A thrombogenic copper coil is advanced to either femoral artery via the left carotid artery. By virtue of the favorable anatomical angles of attachment, a hollow polyurethane catheter advanced down the left carotid artery nearly always enters the descending aorta, and with further advancement, either femoral artery without fluoroscopic guidance. A flexible, teflon-coated guidewire is then inserted through the hollow catheter and the latter is removed. A copper coil is then slipped over the guidewire and advanced to the femoral artery (see inset). Femoral artery flow velocity is measured directly and continuously with a Doppler flow probe placed just proximal to the thrombogenic coil and distal to a prominent sidebranch, which is left patent to dissipate any dead space between the coil and the next proximal sidebranch. Femoral artery blood flow declines progressively to total occlusion over the next 10–12 mm after coil insertion.

2. The mean protein content [mg] of thrombotic material in each dosage group as compared to the vehicle control is determined. Percent change in protein content is calculated relative to control. 3. Statistical significance is assessed by means of the unpaired Student’s t-test. 2.8.2. Eversion Graft-Induced Thrombosis 2.8.2.1. Purpose and Rationale

The eversion graft model of thrombosis in the rabbit artery was first described by Hergrueter et al. (126) and later modified by

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Jang et al. (127, 128) and Gold et al. (15). A 4- to 6-mm segment of the rabbit femoral or the dog left circumflex artery is excised, everted, and then re-implanted into the vessel by end-toend anastomosis. After restoration of blood flow, a platelet-rich occlusive thrombus forms rapidly leading to complete occlusion of the vessel. The rabbit model described here uses a carotid graft inserted into the femoral graft to avoid vasoconstriction, which often occurs in the inverted femoral segments. 2.8.2.2. Procedure

2.8.2.3. Evaluation

In anaesthetized New Zealand white rabbits, the right carotid artery is exposed. After double ligation, a 3-mm segment of the artery is excised, everted, and immersed in pre-warmed (37◦ C) isotonic saline. The right femoral artery is exposed and occluded by means of a double occluder (2-cm distance). The femoral artery is transected and the everted graft from the carotid artery is inserted by end-to-end anastomosis using 12 sutures and 9-0 nylon (Prolene; Ethicon, Norderstedt, Germany) under a surgical microscope (Wild M650; Leitz, Heerbrugg, Switzerland). Perfusion of the graft is measured by means of an ultrasonic flow meter (Model T106; Transonic, Ithaca, NY, USA). The flow probe is positioned 2 cm distal from the graft. After a stabilization period of 15 min, the test substance is administered i.v. through the catheterized right jugular vein. Ten minutes after administration of the test compound, the vessel clamps are released and blood flow is monitored by the flow meter for 120 min. Arterial blood is collected from the left carotid artery at baseline (immediately before administration of test compound), and 10, 60, and 120 min after administration. 1. Time until occlusion = time between restoration of vessel blood flow and occlusion of the vessel, as indicated by a flow of less than 3.0 ml/min. 2. Patency = time during which perfusion of the graft is measured relative to an observation period of 120 min after administration of test compound. 3. Time until occlusion and patency are expressed as median and inter-quartile range/2 (IQR/2). Significant differences (P<0.05) are calculated by the nonparametric Kruskal–Wallis test.

2.8.2.4. Critical Assessment of the Method

The eversion graft is very thrombogenic, although technically difficult and time consuming. The resultant deep occlusive thrombi can be prevented only by intra-arterial administration of thrombolytics or aggressive antithrombotic treatments, such as high doses of recombinant hirudin or PEG-hirudin. Because the initiating surface is a non-endothelial tissue containing tissue factor and collagen, both the coagulation and the blood platelet systems are activated.

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2.8.2.5. Modifications of the Method

Gold et al. (15) described a modification of this model in partially obstructed left circumflexed coronary arteries of thoracotomized dogs. The combination of reduced blood flow due to the constrictor and an abnormal non-endothelial surface produces total thrombotic occlusion within 5 min. Models that use a catheter to induce vessel wall damage of both arteries and veins have been reported (63, 129–131). Such models in the arterial system mimic potential injuries induced by angioplasty. In these models, the endothelium is damaged either by rubbing the catheter across the luminal surface of the vessel or by air desiccation. Inflation of the balloon and the induction of partial stasis in the area of damage produce additional injury. By this procedure, vessel wall collagen, elastic tissues, and tissue thromboplastin are exposed to the circulating blood. Such models are typically carried out in rabbits or larger animals due to size considerations for both the vessel and the catheter. In these models, the formation of thrombi has been detected in a number of ways. Measurement of flow by a distally placed flow meter has been reported (130). A decrease in vessel temperature measured distally to the site of injury is reflective of a decrease in blood flow through the segment and the formation of a thrombus. Deposition of radiolabeled platelets at the site of injury and measurement of thrombus wet weight have also been used. Platelets appear to play an important role in the formation of thrombi at sites where the endothelium is damaged (132). Platelets may also play a key role in the initiation of the restenotic process following angioplasty (133). These models, therefore, provide the opportunity to assess the pharmacologic effects of agents capable of modulating either acute platelet function or the coagulation system that may be useful as adjunctive treatments in angioplasty. It has been demonstrated that both the platelet and the clotting systems are activated by arterial intervention (133, 134) and with this model, it has been shown that heparin and hirudin are both capable of inhibiting initial thrombosis. In addition, these models have also been used to assess the inhibition of re-thrombosis following lysis of the initial clot (130).

2.8.3. Arteriovenous (AV) Shunt Thrombosis 2.8.3.1. Purpose and Rationale

A method for the direct observation of extracorporeal thrombus formation was introduced by Rowntree and Shionoya (135). Very early studies using this model provided evidence that anticoagulants like heparin and hirudin inhibit thrombus development in AV shunts. Today, AV shunt thrombosis models are often used to evaluate the antithrombotic potential of new compounds in different species including rabbits (136), rats

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(137), pigs (138), dogs and cats (139), and non-human primates (140). 2.8.3.2. Procedure

Rats are anaesthetized and fixed in a supine position on a temperature-controlled heating plate to maintain body temperature. The left carotid artery and the right jugular vein are catheterized with short polyethylene catheters. The catheters are filled with isotonic saline solution and clamped. The two ends of the catheters are connected with a 2-cm glass capillary with an internal diameter of 1 mm. This glass capillary provides the thrombogenic surface. At a defined time after administration of test compound, the clamps that are occluding the AV shunt are opened. Patency of the shunt is measured indirectly using a NiCrNithermocouple fixed distal to the glass capillary. When blood is flowing, the temperature rises from room temperature to body temperature. By comparison, decreased temperature indicates the formation of an occluding thrombus. Temperature is measured continuously over 30 min after the opening of the shunt.

2.8.3.3. Critical Assessment of the Method

It has been shown by Best et al. (139) that thrombi formed in the AV shunt are to a great extent white arterial thrombi. This might be due to the high pressure and shear rate inside the shunts, causing thrombi to be more arterial in character (14).

2.8.4. Thread-Induced Venous Thrombosis 2.8.4.1. Purpose and Rationale

Compared to the arterial system, the development of thrombosis models in venous blood vessels tends to be more difficult in terms of reproducibility and variability (14). Complete stasis together with a thrombogenic stimulus (Wessler-type) has been used by a number of investigators to evaluate the effects of test compounds on venous thrombosis. Hollenbach et al. (141) developed a rabbit model of venous thrombosis by introducing cotton threads into the abdominal vena cava of rabbits. The cotton thread serves as a thrombogenic surface, and the thrombus that forms around it reaches a maximum mass after 2–3 h. The prolonged non-occlusive character of thrombogenesis in this model enables studies that focus on the progression of thrombus formation rather than initiation. Thus, conditions more closely resemble the pathophysiology of thrombosis in humans, because blood continues to flow throughout the experiment (14).

2.8.4.2. Procedure

Rabbits weighing 2.5–3.5 kg are anaesthetized by isofluorane inhalation anesthesia, and a polyethylene catheter is inserted into the left carotid artery. A polyethylene tube (PE 240; inner diameter, 1.67 mm) 14 cm in length is filled with isotonic saline, and a copper wire affixed to five fixed cotton threads (6 cm in length) is inserted into the tube (after determination of the net weight of the cotton threads). A laparotomy is performed and the

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vena cava and vena iliac are dissected free from surrounding tissue. The test compound is administered by an intragastric tube for 60 min (depending on the results of ex vivo analysis) prior to initiation of thrombus formation. Blood samples are analyzed 60, 90, 120, 150, and 210 min after oral administration of the test compound. Thrombus formation is induced by inserting the thrombosis catheter into the caval vein via the vena iliaca (7 cm). The copper wire is pushed forward 3 cm to release the cotton threads into the vessel lumen. After thrombus initiation (150 min after initiation), the caval segment containing the cotton threads and the developed thrombus is removed, opened longitudinally, and the content is blotted onto filter paper. After weighing the cotton threads with thrombus, the net dry thread weight is subtracted to determine the corrected thrombus weight. 2.8.4.3. Evaluation

1. Corrected thrombus weight after blotting the thrombus on filter paper and subtraction of the net dry weight of the cotton thread. 2. Mean arterial blood pressure (MAP). 3. aPTT, HepTest, anti-FIIa, and anti-FXa activities.

2.8.4.4. Critical Assessment of the Method

The cotton thread-induced thrombus is composed of fibrin together with tightly aggregated and distorted erythrocytes, similar to human deep vein thrombosis structure. Non-occlusive thrombus formation in this model has been successfully inhibited by heparins, prothrombinase complex inhibitors, and thrombin inhibitors (141, 142).

2.8.4.5. Modifications of the Method

In addition to the originally described method, it is possible to measure blood flow by means of an ultrasonic flow probe attached distal to the position of the cotton threads on the vein.

2.8.5. Thrombus Formation on Superfused Tendon 2.8.5.1. Purpose and Rationale

In all models that include vessel wall damage, blood comes in contact with adhesive proteins of the sub-endothelial matrix, i.e., von Willebrand factor, collagen, fibronectin, laminin, and others. Gryglewski et al. (143) described an in vivo method in which the blood of a non-anesthetized animal is exposed ex vivo to a foreign surface consisting mainly of collagen. The foreign surface is a part of the tendon of another animal species. After superfusion of the tendon, blood is re-circulated to the non-anesthetized animal. This method enables the quantitation of antiplatelet potency based on the formation of platelet thrombi on the surface of the tendon or aortic strips from atherosclerotic rabbits.

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2.8.5.2. Procedure

Blood is withdrawn from the carotid artery of anesthetized and heparinized cats using a roller pump at a speed of 6 ml/min. After a passage through a warmed jacket (37◦ C), blood is separated into two streams, each flowing at a speed of 3 ml/min, that superfuse in parallel two twin strips of the central part of the longitudinally cut rabbit Achilles tendon (30×3 mm). After superfusing the tendon strips, the blood is allowed to drip into collectors and return to the venous system of the animal by gravity through the left jugular vein. The tendon strips are freely suspended in the air. The upper ends are tied to an auxotonic lever of a smooth muscle/heart Harvard transducer; the lower ends are loaded with a weight (1–2 g) to keep the lever with its counterweight in a neutral position. When superfused with blood, the strips become covered with clots, changing the weight of the strips. Weight changes are continuously recorded. After a control period of 30 min, the formed thrombi are gently removed and fixed in formalin for histological examination. The strips are superfused with Tyrode solution, and the animals are injected with antithrombotic (test) compound. After 10 min, blood superfusion is repeated for another 30 min.

2.8.5.3. Evaluation

The ratio of weight increase of the strips after drug treatment relative to before drug treatment is taken as an index of antiaggregatory activity.

2.9. Stasis-Induced Thrombosis (Wessler Model) 2.9.1. Purpose and Rationale

The Wessler model is a classic method of inducing venous thrombosis in animals. Wessler (3, 144–148) combined local venous stasis with hypercoagulability produced by injection of human or dog serum into the systemic circulation of dogs or rabbits. The jugular vein of these animals is occluded by clamps 1 min after injection of the procoagulatory stimulus into the circulation. Within a few minutes after clamping, a red clot is formed in the isolated venous segment. Fareed et al. (149) summarizes the variety of substances that can be used as procoagulatory stimuli in this model. Aronson and Thomas (150) found an inverse correlation between the duration of stasis and the amount of hypercoagulation agent used to produce the clot.

2.9.2. Procedure

Anaesthetized rabbits are fixed in a supine position on a temperature-controlled (37◦ C) heating table. Following cannulation of both carotid arteries (the left in a cranial direction) and the right vena femoralis, segments (2 cm in length) of the two external jugular veins are exposed and isolated between

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two loose sutures. Calcium thromboplastin (0.3 ml/kg) (Sigma; Deisenhofen, Germany, FRG) is administered via the left carotid artery. Meticulous care is taken to maintain a standard injection time of 30 s followed by injection of 0.5 ml of physiological saline within 15 s. Both jugular vein segments are occluded 45 s later by distal and proximal sutures. Stasis is maintained for 30 min. Blood samples are taken immediately before occlusion and 30 s before the end of stasis. After excision, the occluded vessel segments are placed on a soaked sponge and opened by longitudinal incision. 2.9.3. Evaluation

The size of the clots is assessed using a scoring system: 0, blood only; 1, very small clot piece, filling up to 1/4 of the vessel; 2, larger clot, filling up to 1/2 of the vessel; 3, very large clot, filling up to 3/4 of the vessel; 4, a single, large clot that fills the whole vessel. The scores for the left and the right jugular veins are added to arrive at a thrombus size value for each animal. Thrombus weight is also measured after blotting the thrombus on filter paper. Thrombus score is expressed as a median (minimum– maximum). Thrombus weight is expressed as mean ±SEM. For the statistical evaluation of antithrombotic effects, the nonparametric U-test of Mann and Whitney (thrombus score) or Student’s t-test for unpaired samples (thrombus weight) is used. Significance is expressed as P < 0.05.

2.9.4. Critical Assessment of the Method

Because of its static design, Breddin (151) described the Wessler model as the retransformation of an in vitro experiment into a very artificial test situation. One of the major drawbacks of the Wessler model is that it is relatively independent of platelet function and hemodynamic changes that largely influence thrombus formation in vivo. However, the model has been shown to be very useful for evaluating the antithrombotic effects of compounds like heparin and hirudin.

2.9.5. Modifications of the Method

There are a number of different procoagulant agents, such as human serum, Russel viper venom, thromboplastin, thrombin, activated prothrombin complex concentrates, and FXa, that have been used to induce thrombosis in this model (149, 150). The sensitivity and accuracy of the model can be improved by injecting iodinated fibrinogen into the animals before injecting the thrombogenic agent and then measuring specific radioactivity of the clot. A general drawback of the Wessler model is the static nature of venous thrombus development. To overcome this problem, some investigators have developed more dynamic models that incorporate reperfusion of the occluded vessel segments after clot development. Depending on the time of administration of test compound (pre- or post-thrombus initiation), the effect on

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thrombus growth and fibrinolysis can be evaluated. Levi et al. (152) have used this model to assess the effects of a murine monoclonal anti-human PAI-1 antibody, and Biemond et al. (153) compared the effects of thrombin and FXa inhibitors to a lowmolecular weight heparin using a modified Wessler model. Venous reperfusion model. New Zealand white rabbits weighing 2.5 kg are anesthetized with 0.1 ml of atropine, 1.0 mg/kg of diazepam, and 0.3 ml of Hypnorm (Duphar; 10 mg/ml fluanisone and 0.2 ml fentanyl). Anesthesia is maintained with 4 mg/kg i.v. thiopental. The carotid artery is cannulated after exposure through an incision in the neck. The jugular vein is dissected free from tissue and small side branches are ligated over a distance of 2 cm. The vein is clamped proximally and distally to isolate the vein segment. Citrated rabbit blood (from another rabbit) is mixed with 131 I-fibrinogen (approximately 25 mCi/ml), and then 150 μl of radiolabeled blood is aspirated in a 1-ml syringe containing 25 μl of thrombin (3.75 IU) and 45 μl of 0.25 M CaCl2 . An aliquot (200 μl) of the clotting blood is immediately injected into the isolated segment. Thirty minutes after clot injection, the vessel clamps are removed and blood flow is restored. 125 I-fibrinogen (approximately 5 μCi) is injected through the cannula in the carotid artery (in the case of fibrinolysis studies, this is immediately followed by injection of 0.5 mg/kg recombinant t-PA). For each dosage group, four thrombi are analyzed. The extent of thrombolysis is assessed by measuring 131 Ifibrinogen remaining in the clot (relative to initial clot radioactivity). Comparison of 125 I levels in blood and thrombus is a measure of the extent of thrombus growth. Thrombus lysis and extension are monitored 60 or 120 min after thrombus formation and are expressed as a percentage of the initial thrombus volume. Statistical analysis is carried out using variance analysis and the Newman–Keuls test. Statistical significance is expressed as P<0.05. 2.10. Disseminated Intravascular Coagulation (DIC) Model 2.10.1. Purpose and Rationale

Widely used in rats and mice, the DIC model is a model of systemic thrombosis induced by tissue factor, endotoxin (lipopolysaccharide), or FXa (154–156). After systemic administration of a thrombogenic stimulus, studies can be performed with or without mechanical vena caval stasis. When stasis is used, the major parameter is thrombus mass; when stasis is not used, the parameters are primarily fibrin degradation products, fibrinogen, platelet count, prothrombin time (PT), and activated partial thromboplastin time (aPTT). Given the many and varied

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parameters that are measured when stenosis is not used, postexperimental analysis can be time consuming and technically demanding. Although rodents are useful as a primary efficacy model, limitations in drawing multiple blood samples over the course of the experiment and differences in activity of at least some FXa inhibitors in human as compared to rat plasma in vitro require that compounds be further characterized in more advanced in vivo models of thrombosis. 2.11. Microvascular Thrombosis in Trauma Models 2.11.1. Purpose and Rationale

Successful re-plantation of amputated extremities is dependent to a large degree on maintaining the microcirculation. A number of models have been developed in which blood vessels are subjected to crush injury with or without vascular avulsion and subsequent anastomosis (157–159). In the model of Stockmans (159), both femoral veins are dissected from the surrounding tissue. A trauma clamp, which has been adjusted to produce a pressure of 1,500 g/mm2 , is positioned parallel to the long axis of the vein. The anterior wall of the vessel is grasped between the walls of the trauma clamp and the two endothelial surfaces are rubbed together for a period of 30 s as the clamp is rotated. Formation and dissolution of platelet-rich mural thrombi are monitored over a period of 35 min by transillumination of the vessel. By using both femoral veins, the effect of drug therapy can be compared to control in the same animal, minimizing intra-animal variations. The models of Korompilias (158) and Fu (157) examine the formation of arterial thrombosis in rats and rabbits, respectively. In these models, either the rat femoral artery or the rabbit central ear artery is subjected to a standardized crush injury. The vessels are subsequently divided at the midpoint of the crushed area and then anastomosed. Vessel patency is evaluated by milking the vessel at various time points post-anastomosis. These models have been used to demonstrate the effectiveness of topical administration of LMWH in preventing thrombotic occlusion of the vessels. Such models, while effectively mimicking the clinical situation, are limited by the necessity of a high degree of surgical skill to effectively anastomose the crushed arteries.

2.12. Cardiopulmonary Bypass Models 2.12.1. Purpose and Rationale

Cardiopulmonary bypass (CPB) models have been described in baboons (160), swine (161), and dogs (162). In each model, the variables that can affect the hemostatic system such as anesthesia,

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shear stress caused by the CPB pumps, and the exposure of plasma components and blood cells to foreign surfaces (i.e. catheters, oxygenators, etc) are comparable to that observed with human patients. With these models, it is possible to examine the potential usefulness of novel anticoagulants in preventing thrombosis under relatively harsh conditions where both coagulation and platelet function are altered. The effectiveness of direct thrombin inhibitors (160), LMWHs (163), and heparinoids (162) has been compared to standard heparin using this model. Endpoints have included the measurement of plasma anticoagulant levels, histological determination of microthrombi deposition in various organs, formation of blood clots in components of the extracorporeal circuit, and the deposition of radiolabeled platelets in various organs and components of the extracorporeal circuit. These models, therefore, can be used to assess the antithrombotic potential of new agents for use in CPB surgery and also to assess the biocompatibility of components used to maintain extracorporeal circulation. The reader is referred to several detailed protocols and evaluations of this model (3–9, 157–159). 2.13. Extracorporeal Thrombosis Models 2.13.1. Purpose and Rationale

These models employ passing blood over a section of damaged vessel (or other selected substrate) and recording thrombus accumulation on the damaged vessel histologically or by scintigraphic detection of radiolabeled platelets or fibrin (164). This model is interesting because the results can be directly compared to in vivo deep arterial injury model (165) results and to results from a similar extracorporeal model used in humans (166, 167). Dangas et al. (166) used this model to characterize the antithrombotic efficacy of abciximab, a monoclonal antibody-based platelet GPIIb/IIIa inhibitor, after administration to patients undergoing percutaneous coronary intervention. They demonstrated that abciximab reduces both the platelet and the fibrin components of the thrombus, thereby providing further insight into the unique long-term effectiveness of short-term administration of this drug. Ørvim et al. (167) also used this model in humans to evaluate the antithrombotic efficacy of recombinant-tick anticoagulant peptide (rTAP), but instead of evaluating the compound after administration of rTAP to the patient, the drug was mixed with the blood immediately as it flowed into the extracorporeal circuit prior to flowing over the thrombogenic surface. By changing the thrombogenic surface, they were able to determine that rTAP was more effective at inhibiting thrombus formation on a tissue factor-coated surface compared to a collagen-coated surface. These results suggest that optimal antithrombotic efficacy requires an antiplatelet approach along with an anticoagulant.

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Although this model does not completely represent pathological intravascular thrombus formation, the use of this “human model” of thrombosis may be very useful in developing new drugs because it directly evaluates the ex vivo antithrombotic effect of a drug in flowing human blood. 2.14. Experimental Thrombocytopenia or Leucocytopenia 2.14.1. Purpose and Rationale

Intravenous administration of collagen, arachidonic acid, ADP, PAF, or thrombin activates thrombocytes leading to maximal thrombocytopenia within a few minutes. This effect is reinforced by additional injections of epinephrine. Activation of platelets leads to intravascular aggregation and temporary sequestration of aggregates in the lungs and other organs. Depending on the dose of agonist, this experimentally-induced reduction in circulating platelets is reversible within 60 min after induction. Following administration of PAF (or other agonist), leucocytopenia is induced by the addition of epinephrine. This assay is used to test the inhibitory capacity of compounds against thrombocytopenia or leucocytopenia arising from in vivo platelet or leukocyte stimulation.

2.14.2. Procedure

Male guinea pigs (Pirbright White) (300–600 g), male NMRI mice (25–36 g), or Chinchilla rabbits of either sex (2–3 kg) are used. Animals receive test compound or vehicle as a control by oral, i.p., or i.v. administration (Table 2.1). After absorption time (p.o., 60 min; i.p., 30 min; i.v., variable), the marginal vein of the ear of rabbits is cannulated and a thrombocytopenia-inducing substance (i.e., collagen or arachidonic acid) is injected slowly. Blood is collected from the ear artery. Guinea pigs, hamsters, or mice are anesthetized with penR tobarbital sodium (i.p.) and Rompun (i.m.) and placed on ◦ a temperature-controlled table at 37 C. The carotid artery is cannulated for blood withdrawal and the jugular vein is cannulated to administer thrombocytopenia-inducing substance(s), such as collagen+adrenaline, PAF, or thrombin (see Table 2.1). For mice, collagen+adrenaline is injected into a tail vein. Approximately 50–100 μl of blood is collected into potassium-EDTAcoated tubes 1 min before (–1), and 1 and 2 min after injection of the inducer (for guinea pigs and mice), or 5, 10, and 15 min (for rabbits) after inducer. The number of platelets and leukocytes is determined within 1 h of withdrawal in aliquots of 10 μl of whole blood using a microcell counter suitable for blood of various animal species.

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Table 2.1 Experimental thrombocytopenia or leucocytopenia: materials and solutions Substance used to induce thrombocytopenia/leucocytopenia (i.v. administration)

Dose

Rabbits: Arachidonic acid (Sigma)

1 mg/kg

Collagen (Hormonchemie)

30 μg/ml

Mice: Collagen + adrenaline (Hormonchemie)

90 μg/kg + 20 μg/kg

Hamsters: 50 μg/kg + 10 μg/kg

Collagen + adrenaline Guinea pigs: PAF (Paf-acether, Bachem)

0.03–0.04 μg/kg

Thrombin (Hoffman-LaRoche)

60 U/kg

Anesthetics: Pentobarbital sodium (i.p.)

30 mg/kg

Xylazine (i.m.)

8 mg/kg

Urethane (i.p.)

1.5 g/kg

Platelet analyzer: Sysmex microcellcounter F-800

2.14.3. Evaluation

1. The percentage of thrombocytes (or leukocytes) in vehicle control and dosage groups at different time points after injection of the inducer is determined relative to the initial value (before injection). The values for the controls are set as 100%. 2. Percent inhibition of thrombocytopenia (or leucocytopenia) is calculated in each dosage group relative to the control. 3. Statistical significance is evaluated by means of the unpaired Student’s t-test.

2.14.4. Critical Assessment of the Method

The method of collagen + epinephrine-induced thrombocytopenia has been widely used to study the phenotypes of knock-out mice carrying deletions of specific genes implicated in hemostasis/thrombosis. Recent examples are the Gas 6−/− knockout mouse (168) and mice lacking the gene for the Gz small G protein (169). The advantages of this method are that it is a simple experimental procedure, and a small volume of blood is required. In general, application of the method in small animals (mice, hamsters) requires small amounts of test compound. This model is a useful first step in assessing the in vivo antithrombotic efficacy of antiplatelet drugs.

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2.15. Collagenase-Induced Thrombocytopenia 2.15.1. Purpose and Rationale

Intravenous administration of the proteolytic enzyme collagenase leads to the formation of endothelial gaps and exposure of deeper layers of the vessel wall. This type of vascular endothelial injury mainly triggers thrombus formation through the activation of platelets by contact with the basal lamina. As a consequence, thrombocytopenia is induced, which is maximal within 5–10 min following collagenase injection and reversible within 30 min after induction. This model is used as an alternative to the model described above to test the inhibitory capacity of compounds against thrombocytopenia in a model of collagenaseinduced thrombocytopenia in rats.

2.15.2. Procedure

Male Sprague-Dawley rats weighing 260–300 g are used. The animals receive test compound, or vehicle as a control, by oral, i.p., or i.v. administration. After absorption time (i.p., 30 min; p.o., 60 min; i.v., variable), rats are anesthetized with pentobarbital sodium (i.p.) (see Table 2.2). One carotid artery is cannulated for blood withdrawal and one jugular vein is cannulated for injection of inducer. The animals receive an i.v. injection of heparin, and then 20 min later, approximately 100 μl of blood is collected (initial value). Ten minutes later, a thrombocytopenia-inducing substance (collagenase) is administered intravenously. Five, ten, twenty, and thirty minutes after the injection of collagenase, blood samples (approximately 100 μl each) are collected into potassium–EDTA-coated tubes. The number of platelets is determined in 10 μl aliquots of whole blood within 1 h after blood withdrawal using a microcell counter. For additional details of this method, see Volkl and Dierichs (170).

2.15.3. Evaluation

1. The percentage of platelets in vehicle control and dosage groups at the different times following injection of collagen-

Table 2.2 Collagenase-induced thrombocytopenia: materials and solutions Anesthetic: pentobarbital sodium (i.p.)

60 mg/kg

R Heparin (Liquemin ) (i.v.)

500 U/kg

Thrombocytopenia induction: collagenase (i.v.) (E.C. 3.4.24.3; Boehringer Mannheim) Platelet analyzer: Sysmex microcellcounter F-800

10 mg/ml/kg

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ase is determined relative to the initial value for each group. The values for the controls are set as 100%. 2. Percent inhibition of thrombocytopenia is calculated in each dosage group relative to the control. 3. Statistical significance is evaluated by means of the unpaired Student’s t-test. 2.16. Reversible Intravital Aggregation of Platelets 2.16.1. Purpose and Rationale

Isotopic labeling of platelets can be used to monitor platelet aggregation and desegregation in vivo. ADP, PAF, arachidonic acid, thrombin, and collagen are known to induce platelet aggregation. In this model, labeled platelets are continuously monitored in the thoracic (A) and abdominal (B) region of test animals. Administration of aggregation-promoting agents produces an increase in radioactivity in A and a decrease in radioactivity in B. This method assumes that platelets aggregate within the vascular system and accumulate in the pulmonary microvasculature. This in vivo method can be used to evaluate the platelet antiaggregatory properties of test compounds.

2.16.2. Procedure 2.16.2.1. Preparation of Labeled Platelets

Blood is obtained from rats by cardiopuncture. After centrifugation at 240×g for 10 min, platelet-rich plasma (PRP) is transferred to a clean tube and re-suspended in calcium-free Tyrode solution containing 250 ng/ml prostaglandin E1 (PGE1). The suspension is subjected to centrifugation at 640×g for 10 min. The supernatant is discarded and the sediment is suspended by gentle shaking in calcium-free Tyrode solution containing 250 ng/ml PGE1. 51 Cr is added to 1 ml of the platelet suspension. Following a 20-min incubation period at 37◦ C, the suspension is again subjected to centrifugation at 640×g for 10 min. The supernatant is removed and the labeled platelets are re-suspended in 1 ml of calcium-free Tyrode solution containing 250 ng/ml PGE1.

2.16.2.2. Experimental Course

Male Sprague-Dawley or stroke-prone spontaneously hypertensive rats weighing 150–300 g are used. The animals are anaesthetized with pentobarbital sodium (30 mg/kg, i.p.). Following tracheotomy, the vena femoralis is exposed and cannulated. The labeled platelets are administered via the cannula. Circulating platelets are monitored continuously in the thoracic (A) and abdominal (B) region. Counts are collected using a dual channel gamma spectrometer (Nuclear Enterprise 4681) integrated with a microcomputer (AM 9080A). One hour after administration of

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labeled platelets (or when counts in A and B have stabilized), an aggregation-promoting agent (i.e., ADP, PAF, arachidonic acid, thrombin, or collagen) is administered twice by i.v. injection. One hour is allowed to elapse between each i.v. injection. The test compound is administered 2 h after platelet injection concurrently with the fourth administration of the aggregating agent. Thirty minutes (for ADP, PAF, arachidonic acid, or thrombin), or one hour (for collagen) after administration of test compound, another injection of aggregating agent is administered. This injection serves as an additional control or to determine the long-term efficacy of a test compound. 2.16.2.3. Standard Compound

2.16.3. Evaluation

PGI2 (prostacyclin). 1. The microcomputer continuously collects information about aggregation and desegregation of labeled platelets. 2. The following parameters are recorded: A: counts over the thorax B: counts over the abdomen Difference: A–B Ratio: A/B 3. The time course of response is represented by a curve. The area under the curve is calculated using a specific computer software program. 4. Statistical significance is calculated using the Student’s t-test.

2.16.4. Modification of the Method

Oyekan and Botting (171) described a method for monitoring platelet aggregation in vivo in rats using platelets labeled with indium3+ oxine in which the increase in radioactivity in the lung after injection of ADP or collagen was recorded. Smith et al. (172) continuously monitored the intra-thoracic content of intravenously injected 111 indium-labeled platelets in anesthetized guinea pigs using a microcomputer-based system.

3. Animal Models of Bleeding 3.1. Subaqueous Tail Bleeding Time in Rodents 3.1.1. Purpose and Rationale

Blood vessel damage results in the formation of a hemostatic plug, a process that involves several different mechanisms, including vascular spasm, formation of a platelet plug, blood coagulation, and growth of fibrous tissue into the blood clot. A diagnostic parameter for specifically assessing defects of the hemostatic

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system and the influence of drugs on hemostasis is the length of time that it takes for bleeding to stop from a standard incision, the so-called bleeding time. Bleeding-time measurements in animals are used to evaluate the hemorrhagic properties of antithrombotic drugs. Transection of the tail of a rodent was first established by Döttl and Ripke (173), and today it is commonly used in experimental pharmacology. 3.1.2. Procedure

Anaesthetized rats are fixed in a supine position on a temperaturecontrolled (37◦ C) heated table. Following catheterization of a carotid artery (for measurement of blood pressure) and a jugular vein, test compound is administered. After a defined latency period, the tail of the rat is transected with a razor blade mounted on a self-constructed device at a distance of 4 mm from the tip of the tail. Immediately after transection, the tail is immersed into a bath filled with isotonic saline solution (37◦ C).

3.1.3. Evaluation

The time until bleeding stops is determined within a maximum observation time of 600 s.

3.1.4. Critical Assessment of the Method

There are numerous variables that can influence bleeding time measurements in the rodent, as discussed in detail by Dejana et al. (174), including the position of the tail (horizontal or vertical), the environment (air or saline), temperature, anesthesia, and method of injury (i.e., Simplate method, transection). These variables contribute to differences in results reported for compounds like aspirin and heparin analyzed under different assay conditions (175, 176). Furthermore, it is impossible to transect exactly one blood vessel, because the transected tail region consists of a few major arteries and veins, which mutually interact.

3.2. Arterial Bleeding Time in Mesentery 3.2.1. Purpose and Rationale

Arterial bleeding is induced by micro-puncture of small arteries in the area supplied by the mesenteric artery. Bleeding is arrested in viable blood vessels by the formation of a hemostatic plug due to the aggregation of platelets and fibrin formation. In this test, compounds can be evaluated for their ability to inhibit thrombus formation, and thus prolong arterial bleeding time. This test is used to assess agents that interfere with primary hemostasis in small arteries.

3.2.2. Procedure

Male Sprague-Dawley rats weighing 180–240 g receive test compound, or vehicle as a control, by oral, i.p., or i.v. administration. After absorption time (i.p., 30 min; p.o., 60 min; i.v., variable), animals are anesthetized by i.p. injection of 60 mg/kg pentobarbital sodium. Rats are placed on a temperature-controlled table at 37◦ C.

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The abdomen is opened by a midline incision and the mesentery is lifted to display the mesenteric arteries. The mesentery is draped over a plastic plate and superfused continuously with Tyrode solution maintained at 37◦ C. Bleeding times are determined on small mesenteric arteries (125–250 μm external diameter) at the junction of the mesentery with the intestines. Adipose tissue surrounding the vessel is carefully cut with a surgical blade. Arteries are punctured with a hypodermic needle (25 gauge; 16×5/10 mm). The bleeding time of the mesenteric blood vessels is observed through a microscope at a magnification of 40×. The time in seconds is determined from the time of puncture until bleeding is arrested by the formation of a hemostatic plug. 3.2.3. Evaluation

1. Mean bleeding times [s] are determined for each dosage group (4–6 animals/group, 4–6 punctures per animal) and compared to the respective control. 2. Statistical significance assessed by means of the unpaired Student’s t-test. 3. Prolongation of bleeding time in each dosage group is calculated relative to the vehicle control. For additional details on the method and its use in evaluating various mechanisms or agents, refer to Butler et al. (177), Dejana et al. (174), and Zawilska et al. (178).

3.3. Template Bleeding Time 3.3.1. Purpose and Rationale

Template bleeding time is used to detect abnormalities of primary hemostasis due to deficiencies in the platelet or coagulation system by way of a standardized linear incision in the skin (for human). This method has been modified with the development of a spring-loaded cassette with two disposable blades (Simplate II, Organon Teknika, Durham, NC). These template devices ensure reproducibility in the length and depth of dermal incisions. Forsythe and Willis (179) described a modification that enables the Simplate technique to be used to analyze bleeding time in the oral mucosa of dogs.

3.3.2. Procedure

The dog is positioned in sternal or lateral recumbence. A strip of gauze is tied around the mandible and maxilla as a muzzle. The template device is placed evenly against the buccal mucosa, parallel to the lip margin, and triggered. Simultaneously, a stopwatch is started. Blood flow from the incision is blotted using circular filter paper (Whatman No. 1; Fisher Scientific Co., Clifton, NJ) held directly below, but not touching the wound. The filter paper is changed every 15 s. The end point for each bleeding is determined when the filter paper no longer develops a red crescent.

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3.3.3. Evaluation

The time from triggering the device until blood no longer appears on the filter paper is recorded as the bleeding time. The normal range is 2–4 min.

3.3.4. Critical Assessment of the Method

The template bleeding time varies considerably between laboratories, as well as between species and strains. Therefore, it is important to perform the incisions and the blotting in an identical fashion. Prolonged bleeding times have been recognized in dogs with thrombocytopenia, von Willebrand’s disease and uremia, and in dogs treated with aspirin, anticoagulants, and dextran (179, 180). Brassard and Meyers (181) reported that buccal mucosa bleeding time is sensitive to platelet adhesion and aggregation deficits. Generally, the effects of antithrombotic drugs in bleeding time models in animals do not exactly predict bleeding risks in clinical situations. However, the models allow comparison between drugs with different actions (174, 182).

3.3.5. Modifications of the Method

The Simplate device can also be used to perform incisions in the shaved inner ear of rabbits, taking care to avoid major vessels. Normal bleeding time in anaesthetized rabbits is approximately 100 s (77±4 s, n=20) (Just et al. unpublished data). Klement et al. (180) described an ear bleeding model in anaesthetized rabbits. The shaved ear was immersed in a beaker containing saline at 37◦ C. Five full-thickness cuts were made with a No. 11 Bard-Parker scalpel blade, avoiding major vessels, and the ear was immediately re-immersed in saline. At different times thereafter (5–30 min), aliquots of the saline solution were removed, red cells were sedimented and lysed, and cyanohemoglobin was determined as a measure of blood loss. In this study, hirudin produced more bleeding than standard heparin. A cuticle bleeding time (toenail bleeding time) measurement in dogs has been described by Giles et al. (183). A guillotinetype toenail clipper is used to sever the apex of the nail cuticle. A clean transection of the nail is made just into the quick to produce a free flow of blood. The nail is left to bleed freely. The time until bleeding stops is recorded as the bleeding time. Several nails can be cut at one time to ensure appropriate technique. Normal bleeding time in this model ranges from 2 to 8 min.

4. Genetic Models of Hemostasis and Thrombosis 4.1. Purpose and Rationale

Recent advances in genetics and molecular biology have provided tools that allow scientists to design genetically altered animals

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that are deficient in specific proteins involved in thrombosis and hemostasis (so-called knock-outs or nulls) (184, 185). These animals have been extremely useful for identifying and validating novel targets for therapeutic intervention. That is, by examining the phenotype (e.g., spontaneous bleeding, platelet defects, prolonged bleeding after surgical incision, etc.) of a specific knockout strain, scientists can identify the role of the deleted protein. If the phenotype is favorable (e.g., not lethal), pharmacological agents can be designed to mimic the knockout. More recently, novel genetic-based medical approaches have also benefited greatly from the availability of these models, as discussed below. The following section briefly summarizes some of the major findings in thrombosis and hemostasis using genetically altered mice and concludes with an example of how these models have been used in the drug discovery process. The majority of gene knockouts result in mice that develop normally, are born in the expected Mendelian ratios, and are viable, as defined by the ability to survive to adulthood. Although seemingly normal, these knock-out mice exhibit alterations in hemostatic regulation, especially when challenged. Deletion of FVIII, FIX, von Willebrand factor and β3-integrin (186–189) all result in mice that bleed upon surgical challenge, and despite some minor differences in bleeding susceptibility, these mouse knock-out models mirror the human disease states quite well (hemophilia A, hemophilia B, von Willebrand disease, and Glanzmann’s thrombasthenia, respectively). Deletion of some hemostatic factors results in fragile mice with severe deficiencies in their ability to regulate blood loss. Prenatally, these mice appear to develop normally, but they are unable to survive the perinatal period due to severe hemorrhaging, in most cases due to the trauma of birth. Genetic knockouts have also been useful in dissecting the role of individual signaling proteins in platelet activation. Deletion of β3-integrin (188) or of Gαq (190) results in dramatic impairment of agonist-induced platelet aggregation. Alteration of the protein coding region in the β3-integrin carboxy tail, β3-DiY, at sites that are thought to be phosphorylated upon platelet activation also results in unstable platelet aggregation (191). Deletion of various receptors, such as thromboxane A2, P-selectin, P2Y1, and PAR-3, results in diminished responses to some agonists, while other platelet responses are intact (192–195). Deletion of PAR-3, another thrombin receptor in mice, had little effect on hemostasis, indicating the presence of yet another thrombin receptor in platelets, leading to the identification of PAR-4 (192). Given that knockouts of prothrombotic factors yield mice with bleeding tendencies, it follows that deletion of factors in the fibrinolytic pathway results in increased thrombotic susceptibility in mice. Plasminogen (196, 197), t-PA, urokinase-type plasmino-

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gen activator (u-PA), and combined t-PA/u-PA double knockout (198) result in mice with impaired fibrinolysis, susceptibility to thrombosis, vascular occlusion, and tissue damage due to fibrin deposition. Interestingly, due to fibrin formation in the heart, these mice might serve as good models of myocardial infarction and heart failure caused by thrombosis (199). Intriguingly, mice deficient in PAI-1, the primary inhibitor of plasminogen activator, exhibit no spontaneous bleeding and a greater resistance to venous thrombosis due to a mild fibrinolytic state (200), which suggests that inhibition of PAI-1 might be a promising target for novel antithrombotic agents. In addition to their role in the regulation of hemostasis, several targeted genes are important in embryonic development. For example, deletion of tissue factor (201–203), tissue factor pathway inhibitor (TFPI) (204), or thrombomodulin (205) results in an embryonic lethal phenotype. These and other (206, 207) hemostatic factors also appear to contribute to vascular integrity in the developing embryo. These data suggest that initiation of coagulation and generation of thrombin is important at a critical stage of embryonic development, yet other factors must contribute, since some of these embryos are able to progress and survive to birth. Clearly, genetically altered mice have provided valuable insight into the roles of specific hemostatic factors in physiology and pathophysiology. The results of studies using knock-out mice have provided rationale and impetus for attacking certain targets pharmacologically. Knock-out mice have also provided excellent model systems for studying novel treatments for human diseases. For example, genetically altered animals have provided exceptional systems for the development of gene therapy for hemophilia. Specifically, deletion of FIX, generated by specific deletions in the FIX gene and its promoter, results in mice with a phenotype that mimics the human phenotype of hemophilia B (208). When these mice are treated by adenovirus-mediated transfer of human FIX, the bleeding diathesis is fully corrected (209). Similarly, selectively bred dogs that have a characteristic point mutation in the gene sequence encoding the catalytic domain of FIX also have a severe hemophilia B that is phenotypically similar to the human disease (210). Adeno-associated virus-mediated delivery of the canine FIX gene to these dogs intramuscularly resulted in measurable, therapeutic levels of FIX for up to 17 months (211). Clinically relevant partial recovery of whole blood clotting time and aPTT was also observed over this prolonged period. These data provided support for the first study of adeno-associated virus-mediated FIX gene transfer in humans (212). Preliminary results from clinical studies showed evidence of expression of FIX in three hemophilia patients and also provided favorable safety data to substantiate the use of this

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therapeutic at higher doses. Although it is likely that there are differences between the human disease and animal models of hemophilia (or other diseases), it is clear that these types of experiments can provide pharmacological, pharmacokinetic, and safety data that will be extremely useful in designing and developing approaches for safe clinical trials. Gene therapy for patients with bleeding diatheses is more advanced than gene therapy for thrombotic indications. However, promising preclinical data indicates that local overexpression of thrombomodulin (213) or t-PA (214) inhibits thrombus formation in a rabbit model of arterial thrombosis. Similarly, local gene transfer of TFPI prevented thrombus formation in balloon-injured porcine carotid arteries (215). These and other studies (216) suggest that novel gene therapy approaches will also be effective for thrombotic indications, but these treatments will need to be carefully optimized in terms of pharmacokinetics, safety, and efficacy in laboratory animal studies prior to testing in humans. 4.1.1. Knock-out Mice

4.1.1.1. Factor I (Fibrinogen)

Mice are normal in appearance at birth. Approximately 10% die shortly after birth, and another 40% at 1–2 months after birth due to bleeding and/or failure of pregnancy. Blood fails to clot or support platelet aggregation in vitro (217).

4.1.1.2. Factor II (Prothrombin)

Factor II deletion is a partial embryonic lethal mutation, with a 50% rate of death between embryonic day (E) 9.5 and E11.5. At least 25% survive to term, but suffer from fatal hemorrhage a few days after birth. Factor II is important for maintaining vascular integrity during development and post-natally (218, 219).

4.1.1.3. Factor V

Half of the embryos die at E9–E10, possibly as a result of abnormal yolk sac vasculature. The remaining 50% progress normally to term, but die from massive hemorrhage within 2 h of birth. This defect results in a more severe phenotype in the mouse than in human (207, 218).

4.1.1.4. Factor VII

Mice develop normally but suffer fatal perinatal bleeding (219).

4.1.1.5. Factor VIII

Mice exhibit a mild phenotype as compared to severe hemophilia A in humans. There is no spontaneous bleeding, illness or reduced activity during the first year of life. Blood exhibits residual clotting activity (aPTT) (220).

4.1.1.6. Factor IX

Factor IX coagulant activity (aPTT) associated with wild-type, heterozygous, and homozygous null mice is as follows: +/+, 92%; +/−, 53%; −/−, <5%, respectively. Mice suffer from a bleeding disorder, which has been characterized as extensive bleeding

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after clipping a portion of the tail, with bleeding to death if not cauterized (189, 221). 4.1.1.7. Factor X

Partial embryonic lethal (1/3 of the mice died on E11.5 or E12.5), with fatal neonatal bleeding between perinatal day 5 (P5) and P20 (222).

4.1.1.8. Factor XI

APTT is prolonged in homozygous null (−/−) mice (158–200 s) as compared to wild-type (+/+, 25–34 s) and heterozygous (+/−, 40–61 s) mice. No factor XI activity; knockout does not result in intrauterine death; homozygous null exhibit similar bleeding times as wild-type mice, with a tendency to prolongation (223).

4.1.1.9. TF (Tissue Factor)

Abnormal circulation from yolk sac to embryo at approximately E8.5, leading to embryo wasting and death, most likely reflecting the role of TF in blood vessel development (201–203, 224).

4.1.1.10. TFPI

Lethal, with no survivors beyond the neonatal period. Approximately 60% of mice die between E9.5 and E11.5, with signs of yolk sac hemorrhage (204).

4.1.1.11. Thrombin Receptor (TR)

Approximately 50% of the mice die at E9–E10. Fifty percent survive and become normal adult mice at the gross level, with no bleeding diathesis. Null platelets strongly respond to thrombin, whereas null fibroblasts loose their ability to respond to thrombin, indicating the existence of a second TR (206, 225).

4.1.1.12. Thrombomodulin

The null mutation is an embryonic lethal, with embryos dying before the development of a functional cardiovascular system. Mice die before E9.5 due to growth retardation. Heterozygous mice develop normally without thrombotic complications (199, 205, 226, 227).

4.1.1.13. Protein C

Mice appear to develop normally at the macroscopic level, but exhibit obvious signs of bleeding and thrombosis. Mice do not survive beyond 24 h after delivery. Microvascular thrombosis in the brain and necrosis in the liver is observed. Plasma clottable fibrinogen is undetectable, suggesting fibrinogen depletion and secondary consumptive coagulopathy (228).

4.1.1.14. Plasminogen

Mice exhibit severe spontaneous thrombosis, reduced ovulation and fertility, cachexia and short survival, severe glomerulonephritis, impaired skin healing, and reduced macrophage and keratinocyte migration (196, 197).

4.1.1.15. Alpha2-antiplasmin

Mice exhibit normal fertility, viability, and development; no bleeding disorders; spontaneous lysis of injected clots, which is indicative of enhanced fibrinolytic potential; and a significant

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reduction of renal fibrin deposition after lipopolysaccharide (LPS) administration (229). 4.1.1.16. t-PA

Mice exhibit extensive spontaneous fibrin deposition, severe spontaneous thrombosis, impaired neointima formation, reduced ovulation and fertility, cachexia and short survival, severe glomerulonephritis, and abnormal tissue remodeling (198, 199).

4.1.1.17. PAI-1

Mice exhibit reduced thrombotic incidence, no bleeding, accelerated neointima formation, reduced lung inflammation and reduced atherosclerosis. Detailed studies of PAI-1 knock-out mice have been reported by Carmeliet et al. (200), Eitzman et al. (230), Erickson et al. (231), Kawasaki et al. (212), and Pinsky et al. (232).

4.1.1.18. Vitronectin

Mice exhibit normal development, fertility, and survival; serum is completely deficient in “serum spreading factor” and PAI-1 binding activity. Mice also exhibit delayed arterial and venous thrombus formation (233, 234).

4.1.1.19. Urokinase, u-PA (Urinary-Type Plasminogen Activator)

Single u-PA knock-out mice are viable, fertile, and have a normal life span. Mice occasionally exhibit spontaneous fibrin deposition in normal and inflamed tissue and have a higher incidence of endotoxin-induced thrombosis. Combined t-PA/u-PA-knockout mice survive embryonic development, but exhibit retarded growth, reduced fertility, and shortened life span; spontaneous fibrin deposits are more extensive and occur in more organs (198, 235). Transgenic mice carrying the u-PA gene linked to the albumin enhancer/promoter exhibit spontaneous intestinal and intraabdominal bleeding that is directly related to transgene expression in the liver and elevated plasma u-PA levels. Approximately 50% of the transgenic mice die between 3 and 84 h after birth with severe hypofibrinogenemia and loss of clotting function.

4.1.1.20. UPAR (Urinary-Type Plasminogen Activator Receptor)

Mice are phenotypically normal with attenuated thrombocytopenia and mortality associated with severe malaria (236–239).

4.1.1.21. Gas 6 (Growth Arrest-Specific Gene 6 Product)

Mice are viable, fertile, and appear normal; they do not suffer spontaneous bleeding or thrombosis and have normal tail bleeding times. Platelets fail to aggregate irreversibly to ADP, collagen, or U 46619. Arterial and venous thrombosis is inhibited and mice are protected from fatal thromboembolism after injection of collagen plus epinephrine (168).

4.1.1.22. GPIbα (Glycoprotein Ibα, Part of the GPIb-V–IX Complex)

Mice exhibit bleeding, thrombocytopenia and giant platelets (similar to human Bernard Soulier syndrome) (240).

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4.1.1.23. GPV (Glycoprotein V, Part of the GPIb-V–IX Complex)

Mice exhibit increased thrombin responsiveness; GPV null platelets are normal in size; mice have normal amounts of GP Ib-IX and functional von Willebrand factor (vWF) binding. Null platelets are hyper-responsive to thrombin and have an increased aggregation response and shorter bleeding time, reflecting the activity of GPV as a negative modulator of platelet function (241).

4.1.1.24. GPIIb (Integrin Alpha IIb, Glycoprotein IIb, Part of the GPIIb–IIIa Complex)

Mice have a bleeding disorder similar to Glanzmann thrombasthenia in man. Null platelets fail to bind fibrinogen, aggregate and retract a fibrinogen clot; platelet granules do not contain fibrinogen (242).

4.1.1.25. GPIIIa (Integrin Beta3, Glycoprotein IIIa, Part of the GPIIb–IIIa Complex)

Mice are viable and fertile, but have increased fetal mortality. Mice exhibit features of Glanzmann thrombasthenia in man, i.e., defective platelet aggregation and clot retraction, spontaneous bleeding, prolonged bleeding times, dysfunctional osteoclasts, and the development of osteosclerosis with age (188, 243).

4.1.1.26. GPIIa (Glycoprotein IIa, Integrin β1, Part of the GPIa–IIa Complex)

Integrin β1 null platelets from conditional knock-out mice develop normally, and platelet count is normal; collagen-induced platelet aggregation is delayed but otherwise normal; the tyrosine phosphorylation pattern is normal but phosphorylation is delayed. The bleeding time in the bone marrow of chimeric mice is normal, and there are no major in vivo defects (244).

4.1.1.27. vWF

FVIII levels are strongly reduced due to defective protection by vWF. Mice exhibit highly prolonged bleeding times, hemorrhaging, and spontaneous bleeding. vWF knockout mice have been useful for investigating the role of vWF. Mice exhibit delayed platelet adhesion in ferric chloride-induced arteriolar injury (187, 245).

4.1.1.28. Thromboxane A2 Receptor (TXA2R)

Mice exhibit a mild bleeding disorder and altered vascular responses to TXA2 and arachidonic acid (195).

4.1.1.29. Prostacyclin Receptor (PGI2R)

Mice are viable, fertile, and normotensive, with increased susceptibility to thrombosis and reduced inflammatory and pain responses (246).

4.1.1.30. PECAM (Platelet: Endothelial Cell Adhesion Molecule)

Mice exhibit normal platelet aggregation; Duncan et al. (247) and Mahooti et al. (248) described prolonged bleeding times in PECAM knockout mice.

4.1.1.31. Pallid (Pa)

The pallid mouse, one of 13 hypo-pigment mouse mutants with a storage pool deficiency, is a model of human Herman sky Pudlak syndrome (the beige mouse is a model of Chediak Higashi syndrome). Pallid mice exhibit prolonged bleeding times, pigment dilution, elevated kidney lysosomal enzyme, serum α1 antitrypsin deficiency and abnormal otolith formation. The gene defective in pallid mice encodes the highly charged 172-amino

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acid protein pallidin, which interacts with syntaxin 13, a protein that mediates vesicle docking and fusion (249). 4.1.1.32. Gα(q) (Guanyl Nucleotide Binding Protein Gαq)

Blood from knock-out mice exhibits defective aggregation in response to ADP, TXA2, thrombin, and collagen; shape change is normal (190, 250).

4.1.1.33. Gz (A Member of the Gi Family of G Proteins)

Mice exhibit impaired platelet aggregation in response to epinephrine, resistance to fatal thromboembolism, and exaggerated responses to cocaine. Morphine and antidepressant drugs have a reduced effect in knock-out mice (169).

4.1.1.34. Phospholipase Cγ

Mice are viable and fertile, with decreased numbers of mature B cells, defective B cell and mast cell function, and defective Fcγ receptor signaling, resulting in a loss of collagen-induced platelet aggregation (251).

4.1.1.35. CD39 (Vascular ATP Diphosphohydrolase)

Mice are viable and fertile, with prolonged bleeding times but minimally perturbed coagulation parameters. Mice exhibit reduced platelet interaction with injured mesenteric vasculature in vivo; failure of platelets to aggregate in response to standard agonists in vitro is associated with purinergic P2Y1 receptor desensitization and fibrin deposition at multiple organ sites (252).

4.1.1.36. Protein Kinase, cGMP-Dependent, Type 1

Mice are viable and fertile, unresponsive to cGMP and NO, and defective in vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Mice exhibit increased adhesion and aggregation of platelets in vivo in ischemic/re-perfused mesenteric microcirculation and no compensation by the cAMP kinase system (253).

4.1.1.37. VASP

Mice are viable and fertile and exhibit mild platelet dysfunction with megakaryocyte hyperplasia, increased collagen/thrombin activation, and impaired cyclic nucleotide-mediated inhibition of platelet activation (254, 255).

4.1.1.38. Arachidonate 12-Lipoxygenase (P-12LO)

Platelets exhibit a selective hypersensitivity to ADP, manifested as a marked increase in slope and percent aggregation in ex vivo assays, and increased mortality in an ADP-induced mouse model of thromboembolism (256, 257).

4.1.1.39. Arachidonate 5-Lipoxygenase (P-5LO)

Mice develop normally and are healthy. They exhibit no differences in reaction to endotoxin shock as compared to wild-type mice; however, they are resistant to the lethal effects of shock induced by PAF. Inflammation induced by arachidonic acid in these mice is markedly reduced (256).

4.1.1.40. Thrombopoietin

Thrombopoietin-null and thrombopoietin receptor (c-Mpl)-null mice exhibit a 90% reduction in megakaryocyte and platelet levels.

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However, despite these low platelet levels, mice do not exhibit excessive bleeding. Platelets that are present are morphologically normal and functional, indicating that in vivo, thrombopoietin is required for control of megakaryocyte and platelet number, but not maturation (258).

4.1.1.41. Thrombospondin-1

Mice exhibit normal thrombin-induced platelet aggregation and higher numbers of circulating white blood cells.

Table 2.3 Genetic models of thrombosis and hemostasis Gene target

Viable

Embryonic development/survival

References

Protein C

No

Normal/perinatal death

(228)

Fibrinogen

Yes

Normal/perinatal death

(217)

Fibrinogen-QAGVD

Yes

Normal

(217)

FV

No

Partial embryonic loss/perinatal death

(207)

Coagulation

FVII

Yes

Normal/perinatal death

(219)

FVIII

Yes

Normal

(186)

FIX

Yes

Normal

(189)

FXI

Yes

Normal

(223)

Tissue factor

No

Lethal

(201, 203)

TFPI

No

Lethal

(204)

vWF

Yes

Normal

(187)

Prothrombin

No

Partial embryonic loss/perinatal death

(277, 278)

Yes

Normal/growth retardation

(198)

Fibrinolytic u-PA and t-PA uPAR

Yes

Normal

(237, 238)

Plasminogen

Yes

Normal/growth retardation

(196, 197)

PAI

Yes

Normal

(200)

Thrombomodulin

No

Lethal

(205)

Platelet B3

Yes

Normal/partial embryonic loss

(188)

B3-DiYF

Yes

Normal

(191)

P-Selectin

Yes

Normal

(194)

PAR-1

Yes

Normal

(206)

PAR-3

Yes

Normal

(192)

Gαq

Yes

Normal/perinatal death

(190)

TXA2 receptor

Yes

Normal

(195)

P2Y1

Yes

Normal

(193)

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Thrombospondin-1 has been implicated in normal lung homeostasis (258). Mouse knock-out models of virtually all of the known hemostatic factors have been reported, as shown in Table 2.3.

5. Critical Issues in Experimental models 5.1. The Use of Positive Controls

Clearly, there are many antithrombotic agents that can be used to compare and contrast the antithrombotic efficacy and safety of novel agents. The classic antithrombotic agents are heparin, warfarin, and aspirin. However, new, more selective agents such as hirudin, LMWHs, and clopidogrel are commercially available that will either replace or augment these older treatments. Novel antithrombotic agents should certainly be demanded to demonstrate better efficacy than currently available therapy in animal models of thrombosis. This should be demonstrated by performing dose–response experiments that include maximally effective doses of each compound in the model. At the maximally effective dose, parameters such as aPTT, PT, template bleeding time, or other, more sensitive measurements of systemic hypocoagulability or bleeding should be compared. A good example of this approach is a study by Schumacher et al. (259), who compared the antithrombotic efficacy of argatroban and deltaparin in arterial and venous models of thrombosis. Consideration of potency and safety compared to other agents should be taken into account when advancing a drug through the testing funnel. The early in vivo evaluation of compounds that demonstrate acceptable in vitro potency and selectivity requires evaluation of each compound alone in order to demonstrate antithrombotic efficacy. The antithrombotic landscape is becoming complicated by so many agents from which to choose that it will become increasingly difficult to design preclinical experiments that mimic the clinical setting in which poly-antithrombotic therapy is required for optimal efficacy and safety. Consequently, secondary and tertiary preclinical experiments will need to be carefully designed in order to answer these specific, important questions.

5.2. Evaluation of Bleeding Tendency

Although the clinical relevance of animal models of thrombosis has been well-established in terms of efficacy, the preclinical tests for evaluating safety, i.e., bleeding tendency, have not been as predictable. The difficulty in predicting major bleeding, such as intracranial hemorrhage, resulting from antithrombotic

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or thrombolytic therapy stems from the complexity and lack of understanding of the mechanisms involved in this disorder. Predictors of anticoagulant-related intracranial hemorrhage are advanced age, hypertension, intensity and duration of treatment, head trauma, and prior neurologic disease (260, 261). These risk factors are clearly difficult, if not impossible, to simulate in laboratory animals. Consequently, more general tests of anticoagulation and primary hemostasis have been employed. Coagulation assays provide an index of the systemic hypocoagulability of the blood after administration of antithrombotic agents; however, as indicated earlier, the sensitivity and specificity of these assays vary from compound-to-compound, so these assays do not provide a consistent safety measure across all mechanisms of inhibition. Consequently, many laboratories have attempted to develop procedures that provide an indication of bleeding risk by evaluating primary hemostasis after generating controlled incisions in anesthetized animals. Some of the tests used in evaluating FXa inhibitors include template bleeding time, tail transection bleeding time, cuticle bleeding time, and evaluation of clinical parameters such as hemoglobin and hematocrit. Unfortunately, template bleeding tests, even when performed in humans, have not been good predictors of major bleeding events in clinical trials (262–264). However, these tests have been able to demonstrate relative advantages of certain mechanisms and agents over others. For example, hirudin, a direct thrombin inhibitor, appears to have a narrow therapeutic window when used as an adjunct to thrombolysis in clinical trials, producing unacceptable major bleeding when administered at 0.6 mg/kg i.v. bolus, plus 0.2 mg/kg/h (265, 266). When the dose of hirudin was adjusted to avoid major bleeding (0.1 mg/kg and 0.1 mg/kg/h), no significant therapeutic advantage over heparin was observed. If the relative improvement in the ratio between efficacy and bleeding observed preclinically with FXa inhibitors compared to thrombin inhibitors such as hirudin is supported in future clinical trials, this will establish an important safety advantage for FXa inhibitors and provide valuable information for evaluating the safety of new antithrombotic agents in preclinical experiments. 5.3. Selection of Models Based on Species-Dependent Pharmacology/ Physiology

As alluded to earlier, species selection for animal models of disease is often limited by the unique physiology of a particular disease target in different species or by the species specificity of the pharmacological agent for the target. For example, it was discovered relatively early in the development of platelet GPIIb/IIIa antagonists that these compounds were of limited use in rats (267) and that there was a dramatic species-dependent variation in the response of platelets to GPIIb/IIIa antagonists (268–270). This discovery led to the widespread use of larger animals (particularly dogs, whose platelet response to GPIIb/IIIa

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antagonists resembles humans) in the evaluation of GPIIb/IIIa antagonists. Of course the larger animals required more compound for evaluation, which created a resource problem for medicinal chemists. This was especially problematic for companies that generated compounds by combinatorial parallel synthetic chemistry in which many compounds can be made, but usually in very small quantities. However, some pharmacologists devised clever experiments that partially overcame this problem. Cook et al. (271) administered a GPIIb/IIIa antagonist orally and intravenously to rats and then mixed platelet-rich plasma from the treated rats with platelet-rich plasma from untreated dogs. The mixture was then evaluated in an agonist-induced platelet aggregation assay and the resulting inhibition of canine platelet aggregation (rat platelets were relatively unresponsive to this GPIIb/IIIa antagonist) was due to the drug present in the plasma obtained from the rat. Using this method, only a small amount of drug is required to determine the relative bioavailability in rats. However, the animal models chosen for efficacy in that report (guinea pigs and dogs) were selected based on their favorable platelet response to the GPIIb/IIIa antagonist. Similarly for inhibitors of FXa, there are significant variations in the activity of certain compounds against FXa purified from plasma of different species and in plasma-based clotting assays using plasma from different species. DX-9065 is much more potent against human FXa (Ki=78 nM) than against rabbit (Ki=102 nM) and rat (Ki=1980 nM) FXa. Likewise, in the PT assay, DX-9065a was very potent in human plasma (concentration required to double PT, PT×2, was 0.52 μM) and in squirrel monkey plasma (PT×2 = 0.46 μM), but was much less potent in rabbit, dog, and rat plasma (PT×2 = 1.5, 6.5, and 22.2 μM, respectively). Other FXa inhibitors have also demonstrated these species-dependent differences in activity (272–274). Regardless, the investigator must be aware of these differences so that appropriate human doses can be extrapolated from the laboratory animal studies. Although in many cases the exact mechanism for the speciesdependent differences in response to certain therapeutic agents remains unclear, these differences must be examined to determine the appropriate species to be used for preclinical pharmacological evaluation of each agent. This evaluation can routinely be performed by in vitro coagulation or platelet aggregation tests prior to evaluation in animal models. 5.4. Selection of Models Based on Pharmacokinetics

Much debate surrounds the issue as to which species most resembles humans in terms of gastrointestinal absorption, clearance, and metabolism of therapeutic agents. Differences in gastrointestinal anatomy, physiology, and biochemistry between humans and commonly used laboratory animals suggest that no single

Preclinical animal model

Rabbit pulmonary artery thrombosis

Canine coronary cyclic flow reduction (44)

Canine coronary cyclic flow reduction (44)

TPA-induced coronary thrombolysis

Canine coronary cyclic flow reduction (44)

Rabbit jugular vein thrombus growth

Canine coronary artery electrolytic injury (TPA-induced thrombolysis)

Compound

Recombinant t-PA (Activase)

Abciximab (ReoPro)

Tirofiban (Aggrestat)

Eptifibitide (Integrilin)

Enoxaparin (Lovenox)

Hirudin (Refludan)

Argatroban Accelerated reperfusion and prevented re-occlusion

Inhibition of thrombus growth compared to standard heparin

Significant inhibition of platelet-dependent thrombosis

Significant improvement in lysis of occlusive thrombus

Significant inhibition of platelet-dependent thrombosis

Significant inhibition of platelet-dependent thrombosis

Lysis of preformed pulmonary thrombus

Preclinical results

Table 2.4 Animal models of thrombosis and their clinical correlates

(288)

(286)

(50)

(81)

(282)

(39)

(279)

Unstable angina

Deep vein thrombosis after total hip replacement

Unstable angina

Acute myocardial infarction– thrombolysis with t-PA

Unstable angina

High-risk coronary angioplasty

Acute myocardial infarction– thrombolysis

Reference Clinical indication

No episodes of MI during drug infusion

Significant decrease in death, myocardial infarction, and need for revascularization at 30 days Significantly decreased rate of DVT

Improvement in incidence and speed of reperfusion

Reduction in death, myocardial infarction, refractory ischemia

Reduction in death, myocardial infarction, refractory ischemia, or unplanned revascularization

Improved recanalization

Clinical result

(289)

(287)

(285)

(284)

(283)

(281)

(280)

Reference In Vivo Models for the Evaluation of Antithrombotics and Thrombolytics 91

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animal can precisely mimic the gastrointestinal characteristics of humans (275). Due to resource issues (mainly compound availability) and animal care and use considerations, small rodents such as rats are usually considered for primary in vivo evaluation of pharmacokinetics for novel agents. However, there is great reservation about moving a compound into clinical trials based on oral bioavailability data derived from rat experiments alone. Usually, larger animals such as dogs or non-human primates, which have similar gastrointestinal morphology compared to humans, are the next step in the evaluation of pharmacokinetics of new agents. The pharmacokinetic characteristics of the FXa inhibitor YM60828 have been studied extensively in a variety of laboratory animals. YM-60828 demonstrated species-dependent pharmacokinetics, with oral bioavailability estimates of approximately 4, 33, 7, and 20% in rats, guinea pigs, beagle dogs, and squirrel monkeys, respectively. Although these results suggest that YM-60828 has somewhat limited bioavailability, evaluating the pharmacokinetic profile of novel agents in a number of species (276) is a wellestablished approach used to aid in identifying compounds for advancement to human testing. That is, acceptable bioavailability in a number of species suggests that a compound will be bioavailable in humans. Which of the laboratory species adequately represents the bioavailability of a specific compound in humans can only be determined after appropriate pharmacokinetic evaluation in humans. Nevertheless, preclinical pharmacokinetic data are important in selecting the appropriate animal model for testing the antithrombotic efficacy of compounds because the ultimate proof-of-concept experiment is to demonstrate efficacy by the intended route of administration.

6. Clinical Relevance of Data Derived from Experimental Models

Animal models of thrombosis have played a crucial role in the discovery and development of a number of compounds that are now successfully being used for the treatment and prevention of thrombotic diseases. Influential preclinical results using novel antithrombotics in a variety of laboratory animal experiments are listed in Table 2.4, along with the early clinical trials and results for each compound. This table intentionally omits many compounds that were tested in animal models of thrombosis, but failed to be successful in clinical trials or, for other reasons, did not become approved drugs. However, these negative outcomes would not have been predicted by animal models of thrombosis because the failures were generally due to other shortcomings of the drugs (e.g., toxicity, narrow therapeutic window, or

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undesirable pharmacokinetics or pharmacodynamics) which are not always clearly presented in the scientific literature due to proprietary restrictions in this highly competitive field. Nonetheless, it is clear that animal models have supplied valuable information for investigators responsible for evaluating these drugs in humans, providing pharmacodynamic, pharmacokinetic, and safety data that can be used to design safe and efficient clinical trials. The reader is referred to a number of detailed reports on the applications of animal models (188, 190–195, 197–213, 215, 216, 237). References 1. Didisheim, P. (1972) Animal models useful in the study of thrombosis and antithrombotic agents Prog Hemost Thromb 1, 165–97. 2. Leadley, R.J., Jr., Chi, L., Rebello, S.S., and Gagnon, A. (2000) Contribution of in vivo models of thrombosis to the discovery and development of novel antithrombotic agents J Pharmacol Toxicol Methods 43, 101–16. 3. Wessler, S., Reimer, S.M., and Sheps, M.C. (1959) Biologic assay of a thrombosisinducing activity in human serum J Appl Physiol 14, 943–6. 4. Meuleman, D.G., Hobbelen, P.M., Van Dinther, T.G., Vogel, G.M., Van Boeckel, C.A., and Moelker, H.C. (1991) Antifactor Xa activity and antithrombotic activity in rats of structural analogues of the minimum antithrombin III binding sequence: discovery of compounds with a longer duration of action than of the natural pentasaccharide Semin Thromb Hemost 17 (Suppl 1), 112–7. 5. Carrie, D., Caranobe, C., Saivin, S., Houin, G., Petitou, M., Lormeau, J.C., Van Boeckel, C., Meuleman, D., and Boneu, B. (1994) Pharmacokinetic and antithrombotic properties of two pentasaccharides with high affinity to antithrombin III in the rabbit: comparison with CY216 Blood 84, 2571–7. 6. Walenga, J.M., Petitou, M., Lormeau, J.C., Samama, M., Fareed, J., and Choay, J. (1987) Antithrombotic activity of a synthetic heparin pentasaccharide in a rabbit stasis thrombosis model using different thrombogenic challenges Thromb Res 46, 187–98. 7. Vlasuk, G.P., Ramjit, D., Fujita, T., Dunwiddie, C.T., Nutt, E.M., Smith, D.E., and Shebuski, R.J. (1991) Comparison of the in vivo anticoagulant properties of standard heparin and the highly selective factor Xa inhibitors antistasin and tick anticoagulant

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Chapter 3 Heparin and Low-Molecular Weight Heparins in Thrombosis and Beyond Shaker A. Mousa Abstract Heparin and its improved version, low-molecular weight heparin (LMWH), are known to exert polypharmacological effects at various levels. Early studies focused on the plasma anti-Xa and anti-IIa pharmacodynamics of different LMWHs. Other important pharmacodynamic parameters for heparin and LMWH, including effects on vascular tissue factor pathway inhibitor (TFPI) release, inhibition of inflammation through NFκB, inhibition of key matrix-degrading enzymes, selectin modulation, inhibition of platelet–cancer cell interactions, and inflammatory cell adhesion, help explain the diverse clinical impact of this class of agents in thrombosis and beyond. Key words: Heparin, low-molecular weight heparin, tissue factor pathway inhibitor, angiogenesis, tumor growth, metastasis, asthma, inflammation.

1. Introduction Heparin was discovered in 1916 by Jay McLean and William Henry Howell, but did not enter clinical trials until 1935 (1, 2). It was originally isolated from canine liver cells, hence its name (hepar, Greek for “liver”). Howell isolated a watersoluble polysaccharide anticoagulant which was also termed heparin, although it was distinct from the phosphatide preparations previously isolated. The first human trials of heparin began in 1935, and by 1937 it was clear that Connaught’s heparin was a safe, readily available, and effective blood anticoagulant (3). Current commercial heparin is obtained from porcine intestine and is produced by basophils and mast cells. Heparin acts S.A. Mousa (ed.), Anticoagulants, Antiplatelets, and Thrombolytics, Methods in Molecular Biology 663, DOI 10.1007/978-1-60761-803-4_3, © Springer Science+Business Media, LLC 2003, 2010

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as an anticoagulant, preventing the formation of clots and the extension of existing clots within the blood, as well as accelerates thrombolysis when used with standard thrombolytics (4, 5). In recent years, results of clinical studies have clarified the potential and shortcomings of anticoagulant therapy in the prevention and treatment of thromboembolic disorders. The discovery and introduction of heparin derivatives, such as the low-molecular weight heparins (LMWHs), have increased clinical options for the management of thromboembolic disorders while enhancing the safety of therapy. In the United States, LMWHs are currently approved for the prophylaxis and treatment of deep vein thrombosis (DVT), but the use of LMWH is being expanded for additional indications, such as the management of unstable angina and non-Q-wave myocardial infarction (6–9). In addition to its approved uses, LMWH is currently being tested for several newer indications, namely inflammatory diseases and cancer. Additional pharmacological studies and well-designed clinical trials in which multiple pharmacokinetic and pharmacodynamic parameters are analyzed will provide much needed data on the unique clinical profile of each member of this class of novel agents. Clinical experience has confirmed the crucial role of heparin/ LMWH in the success of platelet glycoprotein (GP)IIb/IIIa antagonists (e.g., abciximab, tirofiban, and eptafibatide) for various cardiovascular indications, in that these antagonists lack efficacy and are associated with increased incidence of myocardial infarction in the absence of heparin. In addition, clinical trials have demonstrated clinically relevant effects and improved efficacy of LMWH as compared to unfractionated heparin (UFH) on the survival of cancer patients with DVT. Studies from our laboratory have demonstrated a significant role for LMWH and LMWHreleasable tissue factor pathway inhibitor (TFPI) on the regulation of angiogenesis, tumor growth, cancer-mediated inflammation, and tumor metastasis. In fact, the anti-angiogenic effects of LMWH or non-anticoagulant (NA)-LMWH are reversed by anti-TFPI antibodies. Modulation of tissue factor (TF)/VIIa non-coagulant activities by LMWH- or NA-LMWH-releasable TFPI, as well as inhibition of matrix-degrading enzymes and selectins, and the demonstrated anti-inflammatory efficacy of LMWHs could potentially combine to improve clinical outcomes in patients with vascular thrombosis, cancer, and inflammatory disorders. The key reason behind the success of heparin in thrombosis and beyond is its polypharmacological mode of action in preventing and treating diseases that would benefit only slightly from single pharmacological mechanism-based agents. Thromboembolic disorders are multi-factorial, driven by hypercoaguable, hyperactive platelet, pro-inflammatory, dysfunctional endothelial, and pro-angiogenic states. Heparin can effectively modulate all

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Table 3.1 Molecular targets and polypharmacological effects of LMWHs Mode/site of action

Pharmacological targets

ATIII-dependent plasmatic effects

Anti-Xa, Anti-IIa; other coagulation factors

ATIII-independent vascular effects

TFPI, NO, vWF

Modulation of cell adhesion molecules

Selectins (P, L, and E); immunoglobulins (soluble ICAM-1 and VCAM-1)

Fibrinolytic system

t-PA, PAI-1

Inflammatory mediators

TNF-α, IL-6

LMWH, low-molecular weight heparin; ATIII, antithrombin III; TFPI, tissue factor pathway inhibitor; NO, nitric oxide; vWF, vonWillebrand factor; ICAM, inter-cellular adhesion molecule; VCAM, vascular cell adhesion molecule; t-PA, tissue plasminogen activator; PAI-1, plasminogen activator inhibitor-1; TNF, tumor necrosis factor; IL, interleukin.

Table 3.2 Potential utility of heparin beyond anticoagulation Heparin-sensitive disease state

Effects in experimental models

Clinical status

Adult respiratory distress syndrome

Reduces cell activation and accumulation in airways; neutralizes mediators and cytotoxic cell products; improves lung function in animal models

Controlled clinical trials

Allergic rhinitis

As for adult respiratory distress syndrome; it has not been tested in a specific nasal model

Controlled clinical trials

Arthritis

Inhibits cell accumulation, collagen destruction, and angiogenesis As for adult respiratory distress syndrome; it has been shown to improve lung function in experimental models

Anecdotal reports

Cancer

Inhibits tumor growth, metastasis, and angiogenesis; increases survival time in animal models

Anecdotal reports plus recent clinical trials

Inflammatory bowel disease

Inhibits inflammatory cell transport in general; not tested in a specific animal model

Controlled clinical trials

Asthma

Controlled clinical trials

of these components, as well as interactions among them (Table 3.1). Because heparin was discovered over a half century ago, knowledge of the chemical structure and molecular interactions of this fascinating poly-component was limited in early stages of development. Through the efforts of major multidisciplinary groups of researchers and clinicians, it is now well recognized that heparin has multiple modes/sites of action and can be used for multiple indications (Table 3.2). In the not-too-distant future, we may witness the impact of heparin derivatives on the management of a variety of diseases.

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2. Heparin Versus LMWH Heparin is a glycoaminoglycan formed by sulfated oligosaccharides. Variation in the number of polymeric units results in heparins of different molecular weights. LMWH is generated by partial hydrolysis or enzymatic degradation of UFH. In contrast to UFH, LMWHs have a lower affinity for plasma proteins, endothelial cells, and macrophages (10–12). These differences in binding characteristics underlie the pharmacokinetic differences observed between LMWHs and UFH (Table 3.3). The binding of UFH to plasma proteins reduces its anticoagulant activity, which, combined with variations in the concentration of heparinbinding proteins in plasma, results in an unpredictable anticoagulant response.

Table 3.3 Clinical profile of UFH versus LMWH UFH

LMWH

Continuous, IV infusion

bid or qd subcutaneous injection

Primarily administered in hospital

Administered in hospital, office, or home

Usually administered by health-care professionals

Administered by patient, caregiver, or professional

Monitoring and dosing adjustments

No monitoring; fixed or weight-based dosing

Frequent dosing errors

More precise dosing

Risk of thrombocytopenia and osteoporosis

Decreased risk of adverse events

Inexpensive, but not cost-effective

Demonstrated pharmacoeconomic benefits

Requires 5–7 days in the hospital

Requires 0–2 days in the hospital

UFH, unfractionated heparin; LMWH, low-molecular weight heparin; IV, intravenous; bid, twice daily; qd, every day

LMWHs exhibit many advantageous properties, including improved subcutaneous bioavailability, lower protein binding affinity, and longer half-life, as well as less variability in antithrombin (AT)III recognition sites, glycosaminoglycan content, anti-serine protease activity (anti-Xa, anti-IIa, anti-Xa/antiIIa, and anti-other coagulation factors), induction of TFPI release, and vascular endothelial cell (EC) binding kinetics (13– 16). For these reasons, over the last decade, LMWHs have increasingly replaced UFH for the prevention and treatment of venous thromboembolic disorders. Randomized clinical trials have demonstrated that individual LMWHs used at optimized doses are at least as effective as and probably safer than UFH. Convenient once- or twice-daily subcutaneous (SC) dosing regimens that do not require monitoring have also encouraged the

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wide use of LMWHs. It is well established that different LMWHs vary in their physical and chemical properties due to differences in methods of manufacturing. These differences in turn translate into differences in pharmacodynamic and pharmacokinetic characteristics (17). The World Health Organization (WHO) and United States Food and Drug Administration (US FDA) regard different preparations of LMWH as individual drugs that cannot be used interchangeably (17). The bioavailability of LMWHs after intravenous (IV) or SC administration is greater than UFH, ranging from 87 to 98%. The bioavailability of UFH, by contrast, is 15 to 25% after SC administration. LMWHs have a biological half-life (t1/ ) (based 2 on anti-Xa clearance) nearly double that of UFH. The t1/ of the 2 LMWHs enoxaparin, deltaparin, tinzaparin, and others is between 100 and 360 min, depending on the route of administration (IV or SC). Anti-Xa activity persists longer than AT activity, which reflects faster clearance of longer heparin chains (17). LMWH, in doses based on patient weight, does not require monitoring, probably because of the improved bioavailability, longer plasma t1/ , and more predictable anticoagulant response 2 induced by LMWH as compared to UFH when administered SC. Although LMWHs are more expensive than UFH, a pilot study in pediatric patients found that SC administration of LMWH reduced the number of necessary laboratory assays, nursing hours, and phlebotomy time (18). LMWHs are expected to continue to supplant the use of UFH, as programs are developed for new indications and clinician comfort with use of these drugs increases. In addition, as patients and health-care providers recognize the relative simplicity of administration by SC injection, together with the real cost savings and quality-of-life benefits associated with reduced hospital stays, the trend toward outpatient use will also continue.

3. Emerging Links Between Thrombosis and Inflammation: Potential Role of Heparin

There are several lines of evidence of the interplay between activated platelets/leukocytes and the coagulation cascade. The realization of this dynamic interplay led to the elucidation of a cascade of events that begins with the exposure of platelet GPIIb/IIIa receptors in the active state, leading to platelet fibrinogen binding and amplification of platelet aggregate formation. Activated platelets also interact with leukocytes, leading to platelet– leukocyte cohesion and leukocyte activation. Hyperactive platelets provide a surface for the generation of thrombin, a potent platelet

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and leukocyte activator. In addition, there is significant interplay between the coagulation cascade, platelets, and the blood vessel wall during the progression of thromboembolic disorders. Depending on shear level (i.e., venous, or low shear, versus arterial, or high shear), platelet/fibrin proportion and contributions vary. Infection that leads to the initiation of pro-inflammatory stimuli can be a major predisposing factor in the propagation of thromboembolic disorders. Endotoxin liberated from Escherichia coli and other bacteria can induce a pro-inflammatory state, with increased levels of tumor necrosis factor (TNF)-α and other cytokines (Fig. 3.1). This leads to the activation of leukocytes, increased expression of membrane L-selectin, and the shedding of soluble L-selectin, which can serve as a surrogate marker of leukocyte activation. Activation of leukocytes leads to the propagation and generation of TF, which initiates and amplifies the hypercoaguable state, as well as the up-regulation of TNF-α production (Fig. 3.1). The hypercoaguable state and the generation of thrombin activates platelets, leading to the overexpression of platelet membrane P-selectin and the shedding of soluble P-selectin, which can act as a surrogate marker of platelet activation (19). The pro-inflammatory state can also induce EC insult, leading to increased EC membrane expression and shedding of soluble vascular adhesion molecule-1 (VCAM-1), inter-cellular adhesion molecule-1 (ICAM-1), and E-selectin. Emerging links between thrombosis, angiogenesis, and inflammation in vascular, cardiovascular, and inflammatory disorders are shown in Fig. 3.2. A key role of TF/VIIa in angiogenesis, inflammation, and thrombosis is illustrated in Fig. 3.3.

(A) Hemostasis Activation (TED)

Natural Antiinflammatory factors

Pro-inflammatory Stimuli

Pro-coagulants (B) Inflammatory Diseases

Platelet activation Endothelial activation

Natural anticoagulants, Platelet activation inhibitors Endothelial activation inhibitors

Fig. 3.1. Schematic illustration of hemostasis activation in inflammatory diseases and increased inflammation in thromboembolic disorders (TED).

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Cardiovascular Diseases Vascular Disorders Inflammatory Diseases

Fig. 3.2. Emerging links between thrombosis, angiogenesis, and inflammation in vascular, cardiovascular, and inflammatory diseases.

Possible relationships of TF/VIIa to Angiogenesis, Inflammation & Thrombosis Inflammation TF

Angiogenesis

FVIIa

Inflammation

ABP-280

Coagulation and platelet Activation VEGF & other Factors

Signaling Mechanism?

Thrombosis Fig. 3.3. TF/VIIa in angiogenesis, inflammation, and thrombosis: Impact of heparin releasable tissue factor pathway inhibitor (TFPI).

In recent years, several studies have shown that heparin and LMWH have obvious anti-inflammatory activity in addition to their traditional anticoagulant effects (20, 21). In animal models, heparin disaccharides inhibit TNF-α production by macrophages and decrease immune inflammation (22). Heparin accelerates the healing of mucosa in colitis in several clinical studies and has antiinflammatory effects (23–28). Thus, administration of heparin can induce anti-inflammatory as well as anticoagulant effects. Heparin is currently used for the treatment and prevention of thrombotic and thromboembolic conditions like DVT, pulmonary embolism (PE), and crescendo angina (29–31). Heparin activates ATIII to prevent the conversion of fibrinogen to fibrin and accelerates the inhibition of factors XIIa, XIa, IXa, and Xa. Heparin also possesses non-anticoagulant properties, including the ability to modulate various proteases, as well as anti-complement activity and anti-inflammatory actions. Inhaled

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heparin has been shown to reduce early phase asthmatic reactions (see below) and to suppress allergen-induced rises in bronchial hyper-reactivity. Heparin also inhibits the acute cutaneous reaction to allergens. The ubiquitous distribution of heparin in tissue spaces may serve to limit inflammatory responses in tissues where leukocytes accumulate following an inflammatory challenge. In this regard, it is of note that heparin is found in high concentrations in the gastrointestinal tract and the lung (29, 30), two organs that are exposed to the external environment. The anti-inflammatory activity of heparin has been reinforced by positive, although small, clinical trials in patients suffering from a range of inflammatory diseases, including rheumatoid arthritis and bronchial asthma (35, 36). In addition, a number of clinical studies have recently demonstrated the anti-inflammatory activity of heparin in the treatment of inflammatory bowel disease at doses that do not produce anti-hemorrhagic complications (36) (see below). It is now well recognized that different portions of the heparin molecule mediate anti-inflammatory activity. Given that an isolated pentasaccharide sequence retains the ability to inhibit ATIII (37), it is reasonable to expect that we will reach the point at which the anti-inflammatory and anticoagulant activities of heparin can be separated (38). 3.1. Heparin and Asthma

Heparin significantly reduces asthma symptoms within 10 min of administration. This activity of heparin might be related to its ability to prevent the release of histamine from mast cells and interfere with the stimulation of mast cell mediator secretion through blocking internal calcium release. At later time points, there is evidence to suggest that heparin reduces eosinophil recruitment through several different mechanisms, including prevention of mast cell mediator release, or indirectly through downregulation of adhesion molecules on ECs, thus limiting eosinophil migration into the nasal mucosa (32–34). There is also evidence that the heavily anionic character of heparin inactivates plateletactivating factor, a cationic protein with potent chemotactic activity for human eosinophils. Intranasal heparin attenuates the nasal response to an allergic challenge in atopic rhinitic subjects, with no adverse reactions (32–34). Additional studies are needed to fully understand the mechanisms by which heparin mediates antiinflammatory activity and to optimize heparin use in allergic diseases such as rhinitis and asthma.

3.2. Heparin/LMWH and Inflammatory Bowel Diseases

Several uncontrolled studies have pointed to the potential therapeutic benefit of heparin in the clinical management of ulcerative colitis and Crohn’s disease. Although these studies included only a limited number of patients, they demonstrated apparent beneficial effects of heparin with no associated hemorrhagic complications (36, 39–45). Heparin has also been shown to

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prevent macroscopic inflammatory lesions in an animal model of experimental colitis. However, the molecular mechanisms by which heparin attenuates disease symptoms in patients with inflammatory bowel disease (IBD) are unknown. The therapeutic actions of heparin observed in IBD patients likely involve attenuation of inflammatory processes and the hypercoaguable state associated with clinical exacerbation of IBD, which could in turn promote mucosal repair. 3.3. Heparin as an Anti-inflammatory Molecule: Potential Mechanisms

A large body of evidence supports the concept that heparin has anti-inflammatory actions. Heparin modulates some of the pathophysiological effects of endotoxin and TNF-α, such as neutrophil migration, edema formation, pulmonary hypertension, and hypoxemia (20–22, 24, 26, 28). Moreover, heparin has been shown to suppress specific neutrophil functions, such as superoxide generation (25) and chemotaxis in vitro (23, 27), as well as reduce eosinophil migration (25) and diminish vascular permeability (49, 50). One of the proposed mechanisms by which the anti-inflammatory actions of heparin are mediated is the binding of glycosaminoglycan to adhesion molecules expressed on the surface of activated ECs and/or leukocytes. Recent in vitro studies have shown that heparin effectively binds to endothelial P-selectin, but not E-selectin (21, 51), and to L-selectin and CD11b/CD18 expressed on neutrophils (52, 53). Under different experimental and clinical conditions, heparin was found to actively reduce the process of leukocyte recruitment into sites of injury or applied inflammatory stimuli. Salas et al. (46) provided the first in vivo evidence of an anti-migratory mechanism of action of heparin using intravital microscopy. In fact, intravital microscopic techniques have allowed direct observation of inflamed microvascular beds, which helped define the paradigm of white blood cell extravasation. Leukocyte interaction with the endothelium of an inflamed post-capillary venule is initially intermittent and dynamic (cell rolling); it then becomes static (firm adhesion) and ultimately culminates with diapedesis (47). Using the potent cytokine TNF-α to promote this cascade of events in vivo, Salas and colleagues were able to show that heparin down-regulated TNF-α-induced leukocyte rolling, adhesion, and migration into gut tissue without affecting changes in vascular permeability. These data extended and confirmed previous studies in which heparin reduced leukocyte adhesion to vascular ECs in vitro (48) and recruitment of inflammatory cells into other tissues during experimental inflammatory reactions (38). There have been some reports of the effect of heparin on reactive oxygen species (ROS) generation (54) and cytokine secretion (55) by leukocytes in vitro. It was recently demonstrated that heparin, when injected intravenously into normal subjects at a dose of 10,000 IU, inhibits ROS generation by mono-nuclear cells

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(MNC) and polymorphonucleocytes (PMNs) (56). Heparin has also been shown in a series of experiments using N-acetyl heparin to protect the heart from ischemia-reperfusion injury both in vivo and in vitro, independently of its antithrombin actions (57, 58). It has been suggested that this protection might be due to a reduction in complement activation-mediated injury to the heart (57). Since ischemia-reperfusion injury might also be mediated by oxidative damage (59), it is possible that the protective effect of heparin could be due to an inhibition of ROS generation. A reduction in superoxide radical formation by heparin is likely to increase the bioavailability of nitric oxide (NO) for vasodilatation. In fact, heparin has been shown to exert a vasodilatory effect in normal subjects in vivo (60). Increased bioavailability of NO could in turn have the additional beneficial effect of inhibiting leukocyte adhesion to the endothelium, which would inhibit or retard inflammation (61). NO also inhibits the pro-inflammatory transcription factor NF-κB, which plays a central role in triggering and coordinating both innate and adaptive immune responses.

4. Emerging Links Between Thrombosis and Cancer: Potential Role of Heparin

The association between activation of the coagulation system and systemic thrombosis in human cancer has been recognized for over a century, since Trousseau’s original description of migratory thrombophlebitis complicating gastrointestinal malignancy (62). Greater appreciation in recent years of the interdependency of the coagulation system and malignant behavior has led to an understanding of how an activated coagulation system might enhance cancer cell growth (63). A recent Danish study, while not establishing causality or even a biologic association, showed that patients with cancer who developed venous thrombosis during the course of their disease had significantly shorter cancer-related survival than similar patients who remained thrombosis-free (64). Stronger evidence from several studies, including randomized clinical trials, has demonstrated improved cancer-related survival in patients treated with anticoagulants as compared to those not receiving anticoagulants (65–70). Thromboembolic events have been shown to be important predictors of cancer (69). Thus, cancer screening in patients without identifiable risk factors for thrombosis could be helpful for early detection, diagnosis, and management of disease. Thrombin generation and fibrin formation can be detected consistently in patients with malignancy, who are already at increased risk of thromboembolic complications. This is important, because fibrin formation is also involved in the processes

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of tumor spread and metastasis. Activation of blood coagulation in cancer is a complex phenomenon, involving many different hemostatic pathways and numerous interactions of tumor cells with other blood cells, including platelets, monocytes, and ECs. Tumor cells have the capacity to interact with all components of the hemostatic system. They can directly activate the coagulation cascade by producing their own pro-coagulant factors, or they can stimulate the pro-thrombotic properties of other blood cell components. The etiology of thrombosis in malignancy is multi-factorial; mechanisms include the release of pro-coagulants by tumor cells, plus other predisposing factors leading to a hypercoaguable state that is amplified by chemotherapeutic and radio-therapeutic agents (67, 71–75). In fact, unexplained thromboembolism might be an early indicator of the presence of a malignant tumor before signs and symptoms of the tumor itself become obvious. Hemostatic abnormalities occur in a majority of patients with metastatic cancer. These abnormalities can be categorized as follows: increased platelet aggregation and activation; abnormal activation of the coagulation cascade; release of plasminogen activator inhibitor type 1 (PAI-1); and decreased hepatic synthesis of anticoagulant proteins like protein C and ATIII. Activation of the coagulation cascade is mediated through the release of TF and other pro-coagulants from the plasma membranes of tumor cells (67, 73). Increasing evidence suggests that thrombotic episodes might precede the diagnosis of cancer by months or years, thus representing a potential marker for occult malignancy (71). There has been recent emphasis on the potential risk of cancer therapy (both surgery and chemotherapy) in enhancing the risk for thromboembolic disease (72, 75). Postoperative DVT is indeed more frequent in patients who have undergone surgery for malignant diseases than for other disorders. Both chemotherapy and hormone therapy are associated with an increased thrombotic risk, which can be prevented by low-dose oral anticoagulation (76, 77). The pro-coagulant activities of tumor cells have been extensively studied, raising the possibility that a specific tumor pro-coagulant could serve as a novel marker of malignancy. Cancer disturbs those cellular functions that are critical for homeostasis in multi-cellular organisms, namely, growth, differentiation, apoptosis, and tissue integrity. There have been numerous clinical and experimental studies showing that invasion results from cross-talk between cancer cells and host cells (i.e., platelets, myofibroblasts, ECs, and leukocytes, all of which are themselves invasive). In bone metastases, for example, host osteoclasts serve as targets for therapy. Molecular analysis of invasion-associated cellular activities (namely, homotypic and heterotypic cell–cell

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adhesion, cell–matrix interactions and ectopic survival, migration, and proteolysis) has uncovered multiple underlying branched signal transduction pathways and extensive cross-talk between individual pathways. The role of proteolysis in invasion is not limited to breakdown of extracellular matrix, but also cleavage of proinvasive fragments from cell surface GPs. 4.1. Heparin in Platelet–Cancer Cell Adhesion

Activated platelets release angiogenic growth factors and can therefore potentially contribute to tumor angiogenesis (78–80). Growth factors released by platelets include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) (78–80). Platelets have been implicated in tumor biology (81), and serum levels of VEGF correlate with platelet counts during chemotherapy (82). Furthermore, platelet–tumor cell interactions are believed to be important in tumor metastasis. Expression of TF by tumor cells enhances metastasis and angiogenesis and is primarily responsible for tumor-induced thrombin generation and the formation of tumor cell–platelet aggregates. Activated platelets express and release CD40 ligand (CD40L), which induces endothelial TF expression through ligation to CD40. Recent data have shown that in malignancy, increased cellular TF activity induced by CD40 (tumor cell)–CD40L (platelet) interactions enhances intravascular coagulation and hematogenous metastasis (83). Inhibition of experimental metastasis and tumor growth has been demonstrated in animals by thrombocytopenia and antiplatelet therapies (84, 85). There are several classic studies demonstrating that the formation of tumor cell–platelet complexes in the bloodstream is important in facilitating the metastatic process. Metastasis in animal models can be inhibited by heparin, and retrospective analyses of heparin use in human cancer are yielding promising results (87).

4.2. Treatment of Venous thromboembolism in Cancer Patients

A growing body of evidence suggests that a tumor-mediated hypercoagulation state can develop in cancer patients. There is a strong association between cancer and venous thromboembolism (VTE) (Table 3.4), and patients with cancer are at a remarkably higher risk of VTE than patients who are free from malignant disorders and who experience prolonged immobilization from any cause or following surgical interventions. The management of DVT and PE in patients with cancer represents a potential dilemma for clinicians. Co-morbidities, warfarin failure, hampered venous access, and a high bleeding risk are some of the factors that often complicate anticoagulant therapy in these patients. The use of central venous access devices is increasing, but optimal treatment of catheter-related thrombosis remains controversial. UFH is the traditional standard for initial treatment of VTE, but LMWHs have been shown to be

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Table 3.4 Mechanisms of induction of a hypercoaguable state by tumors Malignant tumors promote Tumor cell surface TF expression Macrophage TF expression Expression of cell surface phospholipids that activate coagulation Tumor-mediated platelet activation and accumulation Tumor-induced endothelial cell activation Tumor-mediated monocyte activation Tumor-mediated fibrin generation Modified from Mousa (93) TF, tissue factor.

equally safe and effective in hemodynamically stable patients. For long-term treatment or secondary prophylaxis, vitamin K antagonists remain the mainstay of treatment. However, the inconvenience and narrow therapeutic window of oral anticoagulants make extended therapy unattractive and problematic. As a result, LMWHs are being evaluated as an alternative for long-term therapy (88, 89). The role of inferior vena cava filters in cancer patients is ill defined, but these devices remain the treatment of choice in patients with contraindications for anticoagulant therapy. In cancer patients affected by DVT, treatment with LMWH has been reported to lower mortality to a greater extent than standard heparin therapy. Several clinical studies of various LMWHs, including enoxaparin, deltaparin, certoparin, and tinzaparin, have demonstrated survival benefits as compared to UFH in cancer patients with certain tumor types and at early stages (90–92). The benefits of LMWH and UFH or warfarin have been examined in ovarian, uterine, lung (small and non-small cell), colorectal, and gastric tumors (89–92). The efficacy and safety profile of LMWH has been shown to be superior to UFH or warfarin (90). This increased efficacy of LMWH as compared to UFH could be related to the improved pharmacokinetic properties of LMWH and ease of use in and out of the hospital (93). The improved efficacy and safety of LMWH as compared to warfarin has also been attributed to properties other than its anticoagulant actions and wider therapeutic index (93). These studies raise the intriguing possibility that LMWHs directly or indirectly modify tumor growth progression. There is convincing evidence for an increased incidence of newly diagnosed malignancy among patients with unexplained VTE during the first 6–12 months after the thromboembolic

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event (62, 94–96). In fact, the existence of a positive feedback loop between tumor and clot that magnifies each entity has been demonstrated (93). Tumor fibrin is a consistent feature of tumor stroma and is deposited shortly after tumor cell inoculation (93, 97). Since fibrin is likely to be beneficial to tumor growth, it is possible that the ability of normal or malignant tissue to generate fibrin influences metastasis (97). 4.3. Heparin/LMWH, TFPI, and Anti-neoplastic Effects

Metastasis is the most deleterious event in cancer and leads to mortality in cancer patients (98). Results from animal studies have shown that metastasis can be inhibited by UFH. There have also been clinical reports of survival benefits from UFH and LMWH that go beyond their antithrombotic actions. Evidence from several studies has suggested putative anti-neoplastic mechanisms for heparin and heparin derivatives (Tables 3.5 and 3.6). However, the primary anti-neoplastic mechanisms of heparin remain to be fully defined.

Table 3.5 Effects of heparin in cancer Inhibits

Stimulates

Angiogenesis

Immune system

Proteases

Differentiation and apoptosis

Growth factors

Nitric oxide production

Coagulation factors

TFPI Release

Oncogene expression Free radical generation Inflammation via NFκB Modified from Mousa (93) NFκB, nuclear factor κB; TFPI, tissue factor pathway inhibitor.

In an experimental model of injectable B16 metastatic melanoma, SC injection of tinzaparin before intravenous injection of melanoma cells reduced lung tumor formation in mice (99). Similarly, intravenous injection of TFPI prior to tumor cell injection reduced B16 lung metastasis and abolished tumor cell-induced thrombocytopenia. These results support the potential role of LMWH and releasable TFPI in tumor growth and metastasis (100). However, it is important to note that heparin and LMWH are thought to inhibit tumor metastasis via different mechanisms (101, 102). 4.4. Activation of Coagulation in Cancer

Many cancer patients have hemostatic abnormalities that predispose them to platelet activation and fibrin formation, leading to clinical or sub-clinical thrombosis (103, 104). Cancer itself leads

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Table 3.6 Comparison of heparin and heparin-derived compounds Heparin-derived compound

Comparison to heparin

Biological activity

Non-anticoagulant; non-immunogenic; orally active Plant derived; minimal anticoagulant activity; anti-inflammatory; orally active

Anti-allergy

Phosphomannopentanose sulfate

Potent inhibitor of heparanase activity

Anti-metastatic; anti-angiogenic; anti-inflammatory

Selectively O-desulfated heparin

Lacks anticoagulant activity

Anti-inflammatory; anti-allergy; anti-adhesive

Heparin tetrasaccharide Pentosan polysulfate

Anti-inflammatory; anti-adhesive; anti-metastatic

to thrombosis, which in turn, enhances the metastatic spread of tumor cells. Heparin therapy is effective and safe for thromboprophylaxis, and LMWH works just as well as or better than UFH. Thus, the antithrombotic action of heparin is a potential mechanism by which it can inhibit malignant processes. TF has been implicated in the up-regulation of proangiogenic factors, such as VEGF, by tumor cells. The mechanism involves complex interactions between tumor cells, macrophages, and ECs, leading to TF expression, fibrin formation, and tumor angiogenesis (105). A recent study has suggested that thrombin generation occurs via the extrinsic (TF-dependent) coagulation pathway on cell surfaces and that some chemotherapeutic agents are able to up-regulate TF mRNA and protein expression in cancer cells (106). Activation of the blood coagulation system stimulates the growth and dissemination of cancer cells through multiple mechanisms. Laboratory data on the effects of anticoagulants on various tumors suggest that this treatment approach has considerable potential in some cancers but not others. For example, renal cell carcinoma (RCC) is one of a small number of human tumors in which the tumor cells contain an intact coagulation pathway, leading to thrombin generation and conversion of fibrinogen to fibrin immediately adjacent to viable tumor cells (107). This is also true for melanoma, ovarian cancer, and small cell lung cancer, but not breast, colorectal, or non-small cell lung cancers (108). This is of considerable relevance given that the growth of melanoma and small cell lung cancer is inhibited by anticoagulants, but not the latter three tumor types (65). Based on the unique features of the interaction of RCC with the coagulation system, RCC might also respond to anticoagulation therapy in a similar manner as small cell lung cancer and melanoma. Thus, anticoagulants that act at the level of TF/VIIa level might have improved efficacy

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and safety in inhibiting tumor-associated thrombosis, angiogenesis, and metastasis. See Table 3.6 for a list of the molecular targets and polypharmacological effects of LMWHs. Tumors differ in the nature of their interactions with the coagulation system. In this regard there are two main types of tumors: (1) those that activate the coagulation system directly and (2) those that mediate coagulation activation indirectly via a paracrine mechanism. Tumors in the first group include RCC, melanoma, and ovarian and small cell lung cancers. These tumors overexpress pro-coagulant molecules such as TF and cancer procoagulant. The entire coagulation pathway is assembled on the tumor cell surface, leading to fibrin formation in close proximity to the tumors. This explains in part the occasional finding in RCC of clots emanating from the tumor and extending into the renal vein and inferior vena cava. Tumors in the second group tend to activate systemic coagulation through the release of cytokines [i.e., TNF-α, interleukin (IL)-1β] that in turn stimulate the production of pro-coagulant molecules on the surface of circulating monocytes. Examples of these tumor types include breast, colorectal, and non-small cell lung cancers. Based on these differences in mechanism of coagulation activation, it is reasonable to predict that tumors in the first group would be more likely to respond to anticoagulants that interfere with TF/VIIa than tumors in the second group. In support of this hypothesis, prospective trials have shown that anticoagulants are strongly active in melanoma and small cell lung cancer, but not in breast, colorectal, and non-small cell lung cancers (65, 66, 68, 70, 109). 4.5. Combination Anticoagulant and Antiplatelet Therapies in Cancer

The processes of blood coagulation and generation of new blood vessels play crucial roles in wound healing. Platelets, for example, are the first line of defense during vascular injury and contain at least a dozen promoters of angiogenesis, the secretion of which into the surrounding vasculature can be induced upon platelet activation by thrombin (85). It follows that these processes are also intricately linked within human tumors. Targeting both the coagulation and angiogenesis pathways could result in more potent anti-tumor effects than targeting either pathway alone. Furthermore, elucidation of the TF signaling pathway in tumor cells should provide new insight into the normal cellular biology of TF and TF-mediated signaling in ECs, smooth muscle cells, and fibroblasts. New classes of anticoagulant molecules have recently been developed that selectively target TF and/or the TF/VIIa complex (86, 87, 110), opening the door to a better understanding of this pathway, and providing a rational basis for the development of new agents to prevent and/or reduce angiogenesis-related disorders and tumor-associated thrombosis and modulate the positive feedback loop between thrombosis and cancer (111).

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Based on the key role of fibrin and platelets in tumor survival, angiogenesis, and metastasis (106, 112), several groups have demonstrated a synergistic benefit of combined anticoagulant and antiplatelet therapy at adjusted doses (113).

5. Heparin and Angiogenesis As discussed above, the coagulation system is activated in most cancer patients and plays an important role in tumor biology. It can also contribute substantially to tumor angiogenesis, a process that represents an imbalance in the normal mechanisms that allow organized healing after injury. A steadily growing and increasingly appreciated body of knowledge of the relationship between the coagulation and the angiogenesis systems has important research and clinical implications. Manipulation of these systems could potentially minimize neo-angiogenesis, which is essential for tumor growth, and cancer-associated thromboembolic complications. Angiogenesis is a process that is dependent on the coordinated production of stimulatory (angiogenic) and inhibitory (angiostatic) molecules. Any imbalance in the regulatory circuit that governs this process can potentially lead to the development of a number of angiogenesis-mediated diseases. Angiogenesis is a multistep process of forming new vessels through sprouting from preexisting vessels. It involves activation, adhesion, migration, proliferation, and transmigration of ECs across cell matrices to or from new capillaries and from existing vessels. The combined defects of overproduction of positive regulators of angiogenesis and deficiencies in endogenous angiostatic mediators has been documented in tumor angiogenesis, psoriasis, rheumatoid arthritis, and other neovascularization-mediated disorders (114, 115). In a several experimental studies, LMWH exhibits potent anti-angiogenesis effects, inhibition of tumor growth, and suppression of metastasis (49, 99, 116, 117). In addition, heparin-sensitive endothelial P-selectin has been shown to be involved in metastasis (118). TF, FGF2, VEGF, and IL-8 are pro-angiogenic molecules (105) that can be inhibited by heparin. However, the mechanisms by which heparin counteracts the functions of these factors differ. The natural inhibitor of TF is TFPI. In the presence of heparin, Zhang et al. (119) showed that TFPI activity is enhanced, and the stimulatory effects of TF on angiogenesis are reduced. Chemokines such as IL-8, on the other hand, have positively charged domains (120). The interaction of heparin with these positive domains could mediate the inhibitory effects of heparin on IL-8.

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In addition to angiogenesis, the adhesion of cells in areas away from the primary tumor growth is a key component of metastasis. Selectins and integrins are families of cellular receptors that mediate cell adhesion and are involved in triggering complex signaling cascades following EC activation. Heparin inhibits selectinand integrin-mediated interactions with tumor cells (121), which suggests that tumor cells can function as ligands and activate these cellular pathways. The LMWH tinzaparin, anti-VIIa antibody (anti-VIIa), and recombinant TFPI (r-TFPI) have been shown to modulate angiogenesis-related processes, including in vitro endothelial tube formation and in vivo angiogenesis mediated by angiogenic factors or by cancer cells (117). Tinzaparin, anti-VIIa, and r-TFPI significantly inhibit EC tube formation at comparable levels in a concentration-dependent manner, and all three agents block FGF2-induced angiogenesis in the chick chorioallantoic membrane (CAM) model. Furthermore, significant inhibition of colon or lung carcinoma-induced angiogenesis and tumor growth and tumor regression have been demonstrated with tinzaparin, antiVIIa, and r-TFPI (77). These studies strongly support a significant role for tinzaparin, anti-VIIa, and tinzaparin-releasable TFPI in the regulation of angiogenesis and tumor growth (117).

6. Conclusions Available data from uncontrolled studies suggest heparin is effective in steroid-resistant ulcerative colitis, with 70% complete clinical remission after an average of 4–6 weeks of therapy. The administration of heparin for this indication, however, is not currently justified by the limited available data. LMWH was used in a single trial in patients with steroid-refractory ulcerative colitis, and the results were similar to that of heparin. Given that a prothrombotic state has been described in IBD, and microvascular intestinal occlusion seems to play a role in the pathogenesis of IBD, it is reasonable to suggest that the beneficial effects of heparin in IBD are due in part to its anticoagulant properties. Beyond its well-known anticoagulant activity, heparin also exhibits a broad spectrum of immune-modulatory and anti-inflammatory properties, through inhibition of neutrophil recruitment and reduction of pro-inflammatory cytokines. Heparin and heparin derivatives represent a safe therapeutic option for severe, steroid-resistant ulcerative colitis and other inflammatory disorders, although randomized, controlled trials are needed to confirm these conclusions.

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Many cancer patients reportedly develop a hypercoaguable state with recurrent thrombosis due to the impact of cancer cells, chemotherapy, radiation, immobility, and catheters on activation of the coagulation cascade. Experimental studies have demonstrated that UFH or LMWH interferes with various processes that regulate tumor growth and metastasis, but these results still need to be clinically validated. These processes might include fibrin formation, binding of heparin to angiogenic growth factors such as FGF2 and VEGF, modulation of TF, TFPI release, inhibition of matrix-degrading enzymes, and others. Clinical trials demonstrate a more clinically relevant effect of LMWH as compared to UFH on the survival of cancer patients with DVT, a finding that needs to be validated in a large multi-center trial of cancer patients with tumors of defined type and stage. Recent results have defined the role of LMWH, anti-factor VIIa, and r-TFPI in the modulation of angiogenesis, tumor growth, and tumor metastasis. There is also accumulating evidence that antiplatelet drugs could provide additional benefit in reducing tumor metastasis.

7. Future Perspective The next 5–10 years should see the use of anticoagulants with or without antiplatelet agents such as aspirin becoming more common as a supplement in cancer patients and other patients with a high risk of thrombosis. In addition, heparin derivatives will likely prove to be more effective than standard single mechanism-based agents for cancer prevention and treatment. References 1. Linhardt, R. (1991) Heparin: an important drug enters its seventh decade Chem Indust 2, 45–50. 2. Marcum, J.A. (2000) The origin of the dispute over the discovery of heparin J Hist Med Allied Sci 55, 37–66. 3. Ruty, C. Miracle Blood Lubricant: Connaught and the Story of Heparin, 1928–1937 Health Heritage Research Services, www.healthheritageresearch.com. Originally published in CONNTACT (1996) 9. Accessed on May 5, 2007. 4. Bjork, I., Lindahl, U. (1982) Mechanism of the anticoagulant action of heparin Mol Cell Biochem 48, 161–82. 5. Hirsh, J., and Raschke, R. (2004) Heparin and low-molecular-weight heparin: the seventh ACCP conference on antithrom-

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Chapter 4 Laboratory Methods and Management of Patients with Heparin-Induced Thrombocytopenia Margaret Prechel, Walter P. Jeske, and Jeanine M. Walenga Abstract The clinical effects of heparin are meritorious and heparin remains the anticoagulant of choice for most clinical needs. However, as with any drug, adverse effects exist. Heparin-induced thrombocytopenia (HIT) is an important adverse effect of heparin associated with amputation and death due to thrombosis. Although the diagnosis and treatment of HIT can be difficult and complex, it is critical that patients with HIT be identified as soon as possible to initiate early treatment to avoid thrombosis. Key words: Heparin-induced thrombocytopenia, HIT antibodies, detection, diagnosis, thrombin inhibitors.

1. Pathologic Mechanism of HIT 1.1. Antibody Generation

HIT is an immune-mediated adverse response to heparin treatment (1–3). HIT antibodies are not directed toward heparin specifically, but rather to platelet factor 4 (PF4) in complex with heparin. PF4 is a positively charged protein stored in the alpha granules of platelets. Exposed lysine and arginine residues on the tetrameric PF4 molecule bind to negatively charged heparin molecules (4, 5). This binding exposes cryptic regions within the PF4 molecule creating antigenic neoepitopes (6–8). Multiple PF4 tetramers arrayed in a lattice with several molecules of heparin are highly immunogenic and play a fundamental role in antibody formation (9). Antibodies formed in response to the heparin:PF4 complex (H:PF4) subsequently recognize PF4 bound to cell membranes (10) or other surfaces (11). Antibodies

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to other heparin-binding proteins, such as neutrophil activating protein (NAP2) and interleukin (IL8), have been identified; however, those to PF4 are found in most patients with HIT (12, 13). Due to its smaller molecular size, low-molecular weight heparin (LMWH) has less ability to bind to the PF4 tetramer, alter its configuration, and cause the generation of HIT antibodies. Patients treated with LMWH are 2–3 times less likely to develop HIT antibodies than patients treated with unfractionated heparin (UFH). However, in vitro studies demonstrate that LMWH cross reacts with existing HIT antibodies formed in response to UFH (14). 1.2. Role of HIT Antibodies

HIT antibodies, once formed, become involved in various hemostatic activation processes. Immune complexes of HIT IgG and H:PF4 crosslink platelet FcγIIa receptors, resulting in platelet activation and release of additional PF4. In the presence of heparin there is continued formation of antigenic complexes, initiating a cycle of platelet activation and aggregation, and generation of highly procoagulant platelet microparticles (1, 6, 15, 16). Sustained platelet activation contributes to platelet clearance and thrombin generation that can lead to both thrombocytopenia and HIT-associated thrombosis. Platelets activated by HIT antibodies induce an inflammatory state in which macrophages, monocytes, and neutrophils are activated (15, 17–19). Antibody and leukocyte binding to activated endothelial cells cause the release of tissue factor, plasminogen activator inhibitor (PAI)-1, and cytokines, as well as an upregulation of adhesion molecule expression promoting localized platelet and monocyte binding (17, 20–23). Heparan sulfate on the endothelial cell surface can bind PF4, forming a complex that is recognized by HIT antibodies (10, 23). The inter-relationships of platelets, leukocytes, the endothelium, and the inflammatory state determine the clinical expression of HIT.

1.3. Seroconversion

H:PF4 antibodies are necessary but not sufficient to cause the clinical symptoms of HIT (thrombocytopenia and thrombosis), as many patients who develop HIT antibodies remain asymptomatic. HIT can develop from any heparin exposure, including incidental amounts from heparin flushes or heparin-coated devices. Frequency of seroconversion and development of thrombocytopenia and/or thrombosis associated with HIT are variable and depend on factors such as patient population and presence of comorbid complications (24–26). Development of thrombocytopenia with or without thrombosis is not always proportional to the incidence of seroconversion. While 25–50% of cardiac surgery patients form antibodies, less than 2.0% develop the clinical symptoms of HIT. Among orthopedic patients, 15% can be antibody positive but

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only 5% develop clinical consequences (27). Overall, clinically symptomatic HIT develops in 1.0% of hospital patients receiving heparin in any form (28). 1.4. Additional Immunogenic Potential

The immunogenic potential of heparins is more complex than that of protein-derived drugs such as hirudin, aprotinin, FVIII concentrate, and erythropoietin. Because of their polycomponent nature and their interactions with multiple endogenous proteins (e.g., fibronectin, growth factors, serpins), heparins and related polysaccharides are likely to generate an array of antibodies. Some of these antibodies may modulate the pharmacologic actions of heparin. Thus, apart from the so-called HIT antibodies and their associated thrombosis, patients generating heparin-mediated antibodies may exhibit a therapeutic compromise requiring dose adjustment of the heparin or an alternative approach for anticoagulation.

2. Clinical HIT 2.1. Thrombocytopenia

HIT is typically described as an otherwise unexplained thrombocytopenia starting 4–14 days after administration of heparin. Thrombocytopenia is usually defined as a platelet count <100–150,000×109 /L; however, HIT may also be recognized by a 30–50% drop from the pre-heparin baseline even if the platelet count remains above this threshold. No single definition of thrombocytopenia is appropriate in all clinical situations (29). HIT is particularly difficult to diagnose in patient populations where low platelet counts are typical. In orthopedic and cardiac surgery patients, HIT may be recognized by the pattern of platelet count recovery or by a particular percent decrease compared to the post-surgical platelet level. Patients requiring ventricular assist devices who receive anticoagulation during surgery and for extended post-operative periods of mechanical circulatory support often develop H:PF4 antibodies, but they also have multiple explanations for low platelet counts (30). No guidelines are yet established for HIT in this patient group. In a patient with previous heparin exposure, particularly within the past 120 days, H:PF4 antibodies may already be present and lead to early onset of HIT, even within hours of the next heparin exposure. On the other hand, patients may experience a late drop in platelet count. If the patient is discharged from the hospital within several days after exposure to heparin, the thrombocytopenia will go undetected, and the diagnosis of HIT-associated thrombosis could potentially be missed.

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2.2. Thrombosis

Thrombosis occurs in approximately 35% of HIT patients with thrombocytopenia. Thrombosis can be apparent at the time of HIT diagnosis, others can develop thrombosis within the next 30 days if not treated with a non-heparin anticoagulant. HIT is associated with a wide spectrum of arterial and venous thromboembolic complications (e.g., deep vein thrombosis, pulmonary embolism, myocardial infarction, thrombotic stroke, ischemic limb, vein graft occlusion, skin lesions at injection sites). Mortality among patients with HIT thrombosis is as high as 30%, with 20% of those surviving requiring a limb amputation.

2.3. Scoring Systems

Diagnosis of HIT is based primarily on clinical presentation. Algorithms and diagnostic scoring systems have been proposed to help clinicians evaluate clinical impressions based on the timing and extent of thrombocytopenia and the presence or absence of thrombotic complications or other explanations for low platelet count (31–33). The performance of such risk assessment strategies varies with the experience of the clinician (34). A separate scoring system has been proposed for use in patients following coronary artery bypass surgery (35). This score takes into account the platelet count time course, the time between surgery and the index date and the duration of surgery. In a retrospective study, changes in the clinical score between initial testing and subsequent testing in patients with an initial negative ELISA were predictive of the development of HIT with thrombosis (36). Such scoring systems offer the possibility of focusing lab testing on those patients most likely to develop clinical HIT and to identify those most likely to benefit from alternate anticoagulant therapy. Further prospective clinical studies are needed to determine the usefulness of such scoring systems in guiding HIT management.

3. Laboratory Methods There are two types of laboratory tests for HIT (Table 4.1). Antigen assays detect the presence of immunoglobulins that bind the antigenic neoepitopes exposed in H:PF4 complexes. Functional assays demonstrate platelet activation caused by HIT antibody immune complexes. Each type of test provides unique information and should not be used as the sole basis to diagnose HIT. 3.1. Antigen Assays

Antigen assays for HIT detect antibodies that recognize and bind cryptic PF4 epitopes. Two solid phase enzyme-linked

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Table 4.1 Laboratory tests for the diagnosis of HIT Platelet count monitoring • The frequency of monitoring depends on the patient’s risk for developing HIT • General recommendations: – Monitor platelet count daily for UFH – Monitor platelet count every other day for LMWH ELISA-type assays detect the presence of antibodies to the H:PF4 complex • Highly sensitive but not specific • May not be predictive of clinical HIT (“false positive”) • May not correlate with a positive response in the platelet function assay • Long-term clinical implications of antibody positivity unclear (i.e., patients who do not develop HIT clinical symptoms except for antibody generation) Platelet function assays for HIT antibodies • Good association with HIT thrombocytopenia and thrombosis • Two primary types of assays: – 14 C-serotonin release assay (SRA; uses washed platelets) – Heparin-induced platelet aggregation test (PAT; uses platelet-rich plasma) Each type of laboratory assay for HIT provides unique information Each type of laboratory assay for HIT differs in sensitivity and specificity Knowledgeable use and interpretation of each test are important Multiple testing over several days improves chance of identifying a HIT patient Combined results from ELISA, SRA, and PAT improves chance of identifying a HIT patient Laboratory test results should not be used to guide initial therapeutic decisions, but rather to confirm a clinical diagnosis of HIT to guide future therapy

immunosorbent assays (ELISAs) are commercially available. The capture probe of the Asserachrom ELISA (Stago) is heparin in complex with recombinant human PF4; the GTI HAT ELISA (Genetics Testing Institute) utilizes negatively charged polyvinylsulfonate bound with PF4 from human platelets. Results are expressed as optical density (OD); OD values for positive/negative thresholds are provided by the manufacturers. Because HIT antibodies are polyclonal and they differ in specificity and affinity, it is not surprising that these two ELISA kits detect slightly different cohorts of H:PF4 antibodies (37), where opposite results are obtained in 15% of patient samples (38, 39).

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These ELISA assays utilize a global conjugate reagent capable of detecting IgG, IgA, and IgM type HIT antibodies. ELISAs are now being offered that detect only HIT IgG. This is based on studies which demonstrated that only IgG isotype HIT antibodies activate platelets (40, 41). While the IgG-specific assay is more sensitive for overt HIT where patients have marked thrombocytopenia and thrombosis, the global assay may provide earlier evidence of a progressing immune reaction. It is recommended to also test patient specimens in the presence of high concentration heparin for a more accurate diagnosis of HIT. Addition of high heparin to the ELISA test reaction inhibits HIT antibody binding by removing or blocking access to antigenic PF4 epitopes (2). Thus, true HIT antibodies will not bind in the presence of high heparin, and thus there will be a negative response with high heparin. Antibodies with non-specific binding will have positive OD values in the presence as well as in the absence of high heparin. There is data to indicate that when antibody binding was not inhibited by high heparin, patients were unlikely to have HIT, suggesting that the extra step provides useful diagnostic information (42). Two antigen-based rapid detection HIT antibody assays are also available. The particle gel immunoassay (PaGIA) is an agglutination assay which utilizes polystyrene beads coated with H:PF4 complexes and test serum/plasma incubated in a chamber of an ID-MicroTyping test card (DiaMed, Switzerland). Centrifugation of the test card separates the beads cross-linked by H:PF4 complexes which are identified by visual inspection. A more qualitative assessment can be done by testing serial dilutions of specimen and reporting antibody titer in terms of the highest dilution showing a positive result (43). The particle immunofiltration assay (PIFA) (Akers Biosciences, Thorofare, NJ) uses microparticles coated with PF4 within a self-contained minireactor device. Addition of (non-frozen/thawed) serum containing H:PF4 antibodies will cause matrix formation and trap the microparticles within the chamber membrane. Non-matrixed microparticles, in HIT antibody negative specimens, migrate through the membrane and are detected in the test result window of the device. Clinical experience with these point-of-care devices has been mixed (44–46). 3.2. Functional Assays

The functional tests are bioassays that utilize fresh platelets from a known reactive normal donor. Addition of heparin to platelets/patient serum (PF4 present) allows H:PF4 complexes to form and present the HIT antigen. HIT antibodies in the patient serum bind to the H:PF4 complex and cause platelet activation. The serotonin release assay (SRA) is conducted with platelets that have been incubated with 14 C-radiolabeled serotonin, then washed and resuspended in calcium-containing buffer. Platelets are Fcγ receptor-bearing cells that are activated by IgG immune

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complexes. Platelet activation resulting in granule release is detected by the presence of radioactivity in the incubation supernatant. By measuring background radioactivity and total 14 C-serotonin uptake, the strength of the activation response to HIT IgG is quantified as percent serotonin release. Platelet activation is usually defined as 20% or greater serotonin release. Stronger release activity presumably indicates high affinity or high titer antibody; 50–80% release has been shown to be more specific for HIT patients with thrombocytopenia and/or thrombosis (31). The other commonly used functional test for the clinical laboratory diagnosis of HIT is the platelet aggregation test (PAT) for HIT antibodies. This test utilizes donor platelets (prepared as platelet rich plasma), heparin, and patient serum and is performed on a commercial aggregometer where platelet aggregation is measured by an increase in light transmission. Tests conducted in platelet-rich plasma are considered less sensitive than washed platelet assays; however, patient specimens can test positive by PAT but negative by SRA and vice versa (32, 47). For both the SRA and the PAT, an important control to increase the assay specificity is inclusion of both low (0.1 U/ml) and high (100 U/ml) heparin concentrations. A positive result in a HIT diagnostic assay is platelet activation in the presence of low heparin, but not in the presence of high heparin. An activation assay result is “indeterminate” when a specimen causes platelet activation at both low and high concentrations of heparin, which indicates that the antigenic target is not heparindependent. These specimens may contain pre-formed immune complexes or antibodies such as anti-HLA or anti-platelet glycoprotein antibodies. There is considerable donor-related variability in platelet responsiveness in these functional platelet assays, making it imperative that tests are performed with platelets from known reactive donors (48, 49). Each assay should also include known HIT antibody positive and negative control sera. The functional tests for HIT are highly complex, difficult to standardize, and require careful attention to quality control measures. The most reproducible results are obtained when these assays are conducted in experienced reference laboratories (50). Other platelet function assays include the heparin-induced platelet aggregation (HIPA) assay which utilizes washed platelets and a visual assessment of platelet aggregation over time in a microtiter plate (51). The strength of the activation response in this assay is reflected in the lag time until aggregation is observed. HIT antibody immune complexes can also be detected by ADP release measured by lumi-aggregometry or by flow cytometry where either platelet microparticle formation or annexin binding is detected (16, 52–54).

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3.3. Assay Responses 3.3.1. ELISA Specificity

Many specimens that test positive by ELISAs do not cause platelet activation in the SRA or PAT and are not associated with thrombocytopenia or thrombosis. Thus, the ELISA tests are not very specific for HIT (40). On the other hand, a negative ELISA result can rule-out HIT in patients with a low clinical probability of HIT based on clinical symptoms (55). For patients with a negative ELISA test result but who are clinically suspected of having HIT, it is useful to perform a combination of different types of tests for HIT antibodies and to repeat testing over a period of several days to assure that antibody does not develop.

3.3.2. HIT Positive Patients

The most straightforward interpretation of HIT tests is on symptomatic patients with thrombocytopenia and/or thrombosis during the period 5–14 days post-heparin exposure. When assessment of the platelet count and/or thrombotic symptoms give rise to reasonable suspicion of HIT, a positive lab result can affirm the diagnosis of HIT and justify initiating or maintaining alternative anticoagulant therapy. When clinical suspicion is high, one negative test should not rule-out HIT. Repeat testing or use of an additional type of test is advisable, along with careful surveillance of platelet counts. The interpretation and value of HIT diagnostic tests vary with the timing of the collection of the patient specimen and the clinical status of the patient. Pre-operative testing may be useful in surgical patients with recent heparin exposure or previous history of clinically symptomatic HIT or in patients with inflammatory or malignant complications. Assuming a negative pre-operative antibody result, tests within 1–4 days after heparin exposure provide little information, since H:PF4 antibodies would not yet be apparent. In the clinical setting, both platelet activation and antigen tests are typically reported as either positive or negative. Retrospective studies suggest that the magnitude of a positive response might be helpful in making a diagnosis of HIT. Patients with stronger activation results (50–80% serotonin release) and/or higher antibody titers (OD 1.0–1.2) have a greater likelihood of having clinically symptomatic HIT (56–58). When evaluating weaker assay responses, it is important to remember that results may be different on a repeat, subsequent test, particularly in those patients whose OD value was in the upper portion of the negative range (35, 59, 60). While it is advisable to follow this process for results from the ELISA tests, results from the platelet function tests can be more difficult to interpret day to day due to the variabilities observed with platelet activity.

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3.3.3. Post-operative Patients

Interpretation of test results, particularly in ELISA assays, in patients without clinical symptoms of HIT during the postheparin interval is problematic. Many patients have a positive response but do not develop thrombocytopenia or thrombosis. Other patients may have a negative response, yet develop symptoms and a positive test result on subsequent days (e.g., delayedonset HIT). Thus, in this population, the ELISA test result needs to be interpreted with caution.

3.3.4. Additional Risks Associated with Antibodies

There is a growing body of evidence to suggest that even in the absence of thrombocytopenia, anti-H:PF4 antibody generation is associated with an increased risk of thrombotic events (61). This is most clearly seen in cardiovascular patients who have received heparin for the treatment of acute coronary syndrome or during coronary bypass surgery. In these studies, a significant increase in the incidence of typical endpoints such as death, myocardial infarction, recurrent angina, the need for urgent revascularization, and post-operative length of stay was observed in patients who were antibody positive compared to those who were negative (24, 62–64). This increased risk is more subtle in noncardiac patients such as those undergoing hemodialysis, vascular surgery, or post-surgical deep vein thrombosis (DVT) prophylaxis (65–68). Until there is more information on risks associated with HIT-seropositivity itself, however, routine screening of patients without thrombocytopenia or unexplained thrombosis is not recommended.

4. Clinical Management The management of patients with HIT consists of high clinical awareness, early diagnosis, and early treatment. Once HIT is suspected, there is a necessity for immediate intervention to initiate anticoagulation treatment against the high risk of thrombosis (Table 4.2). Cessation of heparin alone is not sufficient to remove the threat of thrombosis (69). One should not wait for laboratory results to act. Also initial therapeutic decisions should not depend upon a positive laboratory test, but should be based upon clinical findings (i.e., thrombocytopenia and/or new thromboembolic events). It is important, however, to have laboratory confirmation of HIT since patients who have HIT are at great risk for recurrence should they be exposed to heparin in the future (70). In other words, lab tests should not be used to guide initial therapeutic decisions but rather to confirm a clinical diagnosis of HIT and guide future therapy.

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Table 4.2 Anticoagulant treatments for patients with HIT Direct thrombin inhibitors (DTI) • Argatroban and lepirudin are the treatments of choice for management of HIT thrombosis • Bivalirudin is under development • Monitor with aPTT • Each DTI has individual pharmacologic characteristics Danaparoid (FXa inhibitor) • Successfully used for over 10 years for the management of HIT • Monitor platelet counts at onset of administration to check for clinically significant cross-reactivity to HIT antibodies Vitamin K Antagonists • Used for long-term thrombosis management • Follow guidelines to facilitate cross-over between the DTI and the VKA to avoid coumadin-induced necrosis and monitoring issues For interventional cardiology procedures in patients with HIT • Argatroban is approved • Bivalirudin is approved for use in non-HIT patients • Monitor with ACT For cardiac surgery in patients with HIT • Best to use UFH; best that it is more than 3 months since patient last exposed to heparin; test to assure that patient is HIT antibody negative • For patients with HIT antibody titer requiring life-saving surgery, alternative anticoagulant can be used but dosing and monitoring regimens are not optimized Potential other anticoagulant choices (future) • Fondaparinux (FXa inhibitor) is not approved for HIT but has been successfully used in some HIT patients; some issues with antibody formation and cross-reactivity have occurred • Idraparinux • Oral FXa inhibitors • Oral DTIs • Other agents • Drug combinations to provide multiple targeted antithrombotic protection

LMWH is contraindicated in patients with HIT because it has a high rate of interacting with established HIT antibodies. Platelet transfusions are also contraindicated in patients with HIT. 4.1. Direct Thrombin Inhibitors (DTIs)

DTIs are strong anticoagulants that inhibit the high level of thrombin generation in patients with HIT. Because the chemical structures of the DTIs are different from that of heparin, these drugs do not generate HIT antibodies nor do they interact with pre-formed HIT antibodies.

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Treatment with a DTI significantly reduces the risk of thrombosis and thromboembolic complications (new thrombosis, amputation, death) associated with HIT (71–74). Significantly more treated patients remained event free and platelet counts recovered more rapidly in patients receiving DTI treatment. Due to the inherent bleeding risk with all DTIs, it is important to monitor treatment. Particular attention should be given to elderly patients and patients with renal/liver failure. The activated partial thromboplastin time test (aPTT) has been recommended as the monitoring assay. In general DTIs are dosed to a 2–2.5-fold increase in the aPTT. Laboratory tests that use a clotting endpoint, such as fibrinogen and coagulation factor assays, will be affected by a DTI “contaminant” in the patient’s specimen (75, 76). True factor levels can be measured by chromogenic or immunologic based assays which are not affected by the DTI. Although there are many similarities among the DTIs, important differences exist between them (Table 4.3) (70, 77, 78). The different chemical structure of each DTI defines where each drug binds to thrombin, the tightness of the binding, and so on. These characteristics are reflected in the different pharmacokinetic and pharmacodynamic behavior of each drug. 4.1.1. Argatroban

Argatroban has been approved by the health authorities of the United States, Canada, and Europe for both the prophylaxis and the treatment of HIT thrombosis. It is hepatically metabolized and thus the anticoagulant of choice in patients with renal failure (79). Argatroban is administered by continuous infusion at a dose of 1.7–2.0 μg/kg/min for 5–7 days (71, 72). Plasma argatroban levels rapidly decline in about 40 min when drug is discontinued, and coagulation parameters generally return to pretreatment values within 2–4 h (80–82). Repeated exposure to argatroban does not generate antibodies to argatroban, bleeding, or other adverse events (83, 84). Argatroban can be distinguished from other DTIs in that it produces an increase in nitric oxide, which may contribute to its therapeutic efficacy by modulating vascular and cellular function (85). Argatroban has also been approved by the US Food and Drug Administration (FDA) for anticoagulation of HIT patients during percutaneous coronary interventions (PCI) (86). There are reports on the successful use of argatroban anticoagulation in pediatric patients requiring interventional cardiology procedures or extracorporeal life support (87, 88) and for stent implantation in renal arteries (80, 89, 90). Argatroban anticoagulation in PCI (at a reduced dose) used in combination with glycoprotein IIb/IIIa inhibition for PCI is well tolerated with an acceptable bleeding risk (91).

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Table 4.3 Comparison of the pharmacologic responses of the commonly used alternative anticoagulants for the management of HIT thrombosis Argatroban (DTI) • Approved for both prophylaxis and treatment of HIT thrombosis • Approved for use in interventional cardiology procedures in HIT and non-HIT patients • Anticoagulant response rapidly reversed (40–50 min) • Cleared through the liver • Adjust dose in hepatically impaired patients • Affects the INR • Antibodies are not generated against argatroban Lepirudin (DTI; hirudin) • Approved only for treatment of HIT thrombosis • Anticoagulant response slowly reversed (1.3–3 h) • Bleeding tends to be higher with lepirudin than with other DTIs • Cleared through the kidneys • Adjust dose in renally impaired patients • Antibodies are generated in 45% of treated patients; upon re-exposure severe anaphylaxis and death have occurred Danaparoid (FXa inhibitor) • Has been widely used in North America and Europe • Long elimination half-life (25 h) • Can be given either intravenously or subcutaneously • Low bleeding risk • Mainly cleared through the kidneys • Routine monitoring not required; monitoring required in patients with excessively low or high body weight or renal failure (use anti-FXa assay) • Small potential for clinically relevant cross-reactivity with HIT antibodies; platelet counts should be monitored at initiation • Multiple mechanisms of antithrombotic action, including an anti-inflammatory effect

In a more recent study, argatroban was found to effectively reduce new stroke and stroke-associated mortality in patients with HIT without increasing intracranial hemorrhage (92). 4.1.2. Lepirudin

Lepirudin has been approved for the treatment of HIT thrombosis by the health authorities of the United States, Canada, and Europe. It is an irreversible inhibitor of thrombin with an elimination half-life of about 90 min (1.3–3 h). Lepirudin is renally excreted and needs to be used with caution in patients with renal impairment. Intravenous dosing ranges from 0.1–0.4 mg/kg/h, with or without an initial bolus, for 11–14 days in HIT patients

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(73, 74). Bleeding rates tend to be higher with lepirudin than with other DTIs. Exposure to lepirudin results in antibody formation in about half of the treated patients (93–95). These antibodies alter the pharmacokinetics of lepirudin necessitating careful monitoring to avoid bleeding complications. Re-exposure to lepirudin has been linked to at least nine reported cases of severe anaphylaxis with at least five fatal outcomes (96). Lepirudin has been studied in HIT patients undergoing PCI (97), but it has not been approved for this setting. 4.1.3. Bivalirudin

Bivalirudin is a reversible inhibitor of thrombin that is largely renally excreted. Although not approved by the FDA, bivalirudin has been used to anticoagulate patients with HIT thrombosis. Bivalirudin is, however, approved for use in PCI in non-HIT patients (98). Perhaps the greatest obstacle to overcome in the management of patients with HIT is anticoagulation during surgical coronary revascularization or heart valve replacement surgery. For patients with active HIT, anticoagulation with bivalirudin was shown to be feasible in both on-pump (cardiopulmonary bypass, CPB) and off-pump (OPCAB) cardiac surgery (99, 100). However, the use of any DTI in cardiac surgery is associated with inherent risks because there is no antidote for any DTI. Dosing guidelines have not been fully established and bleeding can be excessive, and monitoring the high drug levels is an unresolved issue. Complete efficacy against blood clotting is also a concern.

4.1.4. Oral Thrombin Inhibitors

The oral DTI dabigatran was approved March 2008 for the prevention of venous thromboembolic events in patients who have undergone total hip replacement surgery or total knee replacement surgery in Europe. This drug can be considered for anticoagulation of patients with HIT thrombosis. Although this small molecule, direct acting, thrombin inhibitor has not been studied in HIT, based on its chemical structure, it is not expected to bind PF4 or have any interaction with pre-formed HIT antibodies.

4.2. Factor Xa Inhibitors 4.2.1. Danaparoid

Danaparoid has been used to successfully treat HIT patients for more than 10 years (101–103). Because danaparoid has a low bleeding risk, routine monitoring is not required, except in patients with excessively low or high body weight or renal failure (104). It has a sustained effect and can be given either intravenously or subcutaneously. There is a small potential for clinically relevant cross-reactivity of danaparoid with HIT antibodies

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in patients, so platelet counts should be monitored during the initial phase of treatment (105, 106). A potential advantage of danaparoid over the DTIs is that it has multiple mechanisms of action, including an antiinflammatory effect. Thus in addition to inhibition of the coagulation system, danaparoid may be able to affect other aspects of the pathophysiology of HIT. 4.2.2. Fondaparinux

The synthetic derivative of heparin, fondaparinux, is of very low molecular weight and does not bind to PF4. Thus, it may be a useful alternative anticoagulant for HIT patients. In fact, there are reports of its successful use in several HIT patients (107–110). However, there is also one report of HIT-induced thrombosis associated with fondaparinux treatment (111) and another report of fondaparinux interacting with LMWH-induced HIT antibodies (112). Furthermore, there is evidence that HIT-type antibodies are generated with fondaparinux treatment (113). At this time and until more is known, fondaparinux should be used with caution in patients with HIT.

4.2.3. Idraparinux

Idraparinux is a sister molecule of fondaparinux. Its extended halflife allows for once per week dosing. This agent is in clinical trial for prophylaxis against venous thrombosis. As fondaparinux has showed success in a limited number of patients with HIT, idraparinux may also be considered for use in HIT; however, no reports have been published. Other structurally modified derivatives of fondaparinux, such as non-PF4 binding agents, which are in development, may be of interest in the future.

4.2.4. Oral XaI Inhibitors

Small molecule, direct acting, factor Xa inhibitors that can be orally administered include rivaroxaban and apixaban. Both have been studied in clinical trials for prophylaxis against venous thrombosis following orthopedic surgery (114–117). In September 2008, rivaroxaban was approved in Europe and Canada, and continues to seek approval from the US FDA. Apixaban is awaiting approval in several countries. These agents can be considered for use in the management of thrombosis in patients with HIT because they do not bind PF4, and they should have no interaction with pre-formed HIT antibodies (118). They also should not generate HIT antibodies. Although no clinical information is yet available, if these oral agents are found to be useful for the clinical management of HIT, an advantage would be their application for both acute and long-term treatment.

4.3. Vitamin K Antagonists

For long-term anticoagulation of HIT patients, vitamin K antagonists (VKAs) are used after the treatment period with a DTI. Specific dosing guidelines for the VKA need to be followed to avoid thrombotic complications in patients with HIT (29, 70, 119). VKA can be initiated when the patient is out of the acute phase of HIT (i.e., platelet count on the rise and

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>100×109 /L). It should be started at a low dose (a loading dose should not be used) while the patient is fully anticoagulated with a DTI. The DTI can be tapered off when the INR for the VKA is therapeutic and stable. VKA treatment should continue until platelet counts recover to a stable plateau or longer if clinically warranted. DTIs prolong the PT/INR (75, 120–123). INRs >5 commonly occur with argatroban–warfarin co-therapy but this does not correspond to an effect on coagulation factor levels and bleeding is not enhanced (77, 122). There is a predictable linear effect of argatroban doses up to 2 μg/kg/min on the INR during warfarin co-therapy. This allows for reliable prediction of the level of oral anticoagulation with the VKA (121). To transition from lepirudin to VKA, the dose of lepirudin is first reduced to an aPTT ratio just above 1.5. Lepirudin is then continued for 4–5 days with the VKA and discontinued when the INR is therapeutic (73, 74). 4.4. Further Considerations 4.4.1. Cardiac Surgery

Until a DTI or another anticoagulant is approved for use in cardiac surgery, heparin remains the safest and most effective anticoagulant in this high-risk clinical setting. However, subsequent use of heparin after resolution of HIT can be hazardous particularly within the first 3 months. A brief exposure to heparin during surgery can be considered under compelling circumstances for patients with a history of HIT who have HIT antibodies that are not detectable by a functional platelet assay (70). In this circumstance, standard heparin protocols restricted to the surgery itself can be employed with the use of a DTI or a VKA for postoperative care (70, 124).

4.4.2. Multi-Targeted Treatment

Although DTIs have made a huge advance in the clinical management of patients with HIT thrombosis, there remains an unacceptable rate of morbidity and mortality in this patient population. If one considers the pathophysiology of HIT, it seems obvious that inhibition of thrombin, while important, cannot provide complete antithrombotic management of HIT thrombosis. HIT is associated not only with a hypercoagulable state, but also platelet activation, vascular endothelial dysfunction, and inflammation (leukocyte activation, cytokine up-regulation) (17, 21, 23). HIT is a multi-pathologic condition that may be best treated by multi-targeted therapy. Limited studies suggest that a combination of an inhibitor of thrombin/thrombin generation plus an anti-platelet drug such as GPIIb/IIIa inhibitor (125), not aspirin (126), may be of interest. This combined therapy targets the principle mechanisms related to the pathology of HIT: platelet

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activation and thrombin generation (15, 127). Combinations of a thrombin inhibitor and an FXa inhibitor have also been suggested. 4.4.3. Other Potential Treatment Drugs

Other antithrombotic drugs that can be considered for the management of HIT thrombosis include recombinant thrombomodulin being developed for disseminated intravascular coagulation (DIC), defibrotide, a single-stranded deoxyribonucleic acid derivative used to treat venous occlusive disease, and suledoxide, a heparinoid complex under development for diabetic nephropathy. Although these drugs have not been studied for efficacy or safety in patients with HIT, they have the potential to be useful.

5. Regulatory Considerations An interesting development has recently occurred with the introduction of generic LMWHs. Regulatory bodies are challenged to develop specific guidelines for generic LMWH approval due to the complex nature of these polycomponent biologicals. The US FDA has identified immunogenicity of LMWHs as an important criterion to differentiate LMWHs and to demonstrate the bioequivalence of generic LMWHs to the branded products (128). It remains important to determine how best to demonstrate this equivalence. Demonstration of similar in vitro crossreactivity is likely to be insufficient. Rather, well-designed clinical trials demonstrating a similar propensity to trigger anti-H:PF4 antibodies would be preferred. The immunologic profile of both UFH and LMWH is largely dependent on the heparin components and the presence of other glycosaminoglycans (GAGs) such as dermatans. The starting material for the heparins is heterogeneous, and due to increased demand, the quality issues for heparin are compromised. Therefore, even with UFH the prevalence of HIT antibodies and their composition may vary and should be further investigated. If the proposed US FDA guidelines along with the proposed PCR method (129) are implemented, such differences can be minimized. Because of this, monitoring both UFH and LMWH for HIT is important. Also of concern is the recent issue of heparin contamination by over-sulfated chondroitin sulfate (OSCS) (130, 131). It is known that other highly sulfated GAGs and GAG-like molecules elicit a strong cross-reactivity in in vitro functional assays for HIT. Preliminary data suggest that a similar profile is observed with OSCS. Additionally, the mixture of OSCS and heparin, or the LMWH enoxaparin mixed with OSCS, exhibits a different interaction compared to the heparin alone. Issues related to this

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contaminant are yet to be resolved, and concern exists for potential other contaminants in the future. While published reports have identified OSCS as the primary contaminant, other impurities in the recalled batches of heparin have also been found. Some of these relate to GAGs and are yet to be characterized. Moreover, the molecular profile of OSCS isolated from different contaminated heparins also differs. Some of the recalled batches of LMWHs have been found to contain OSCS with varying molecular weight, suggesting that the parent contaminant in the starting material is depolymerized during the manufacturing process. The presence of a low-molecular weight OSCS in LMWH is of concern as this material binds much more strongly with PF4 and other proteins and could potentially generate additional forms of antibodies whose functions are currently unknown. Therefore, proper monitoring of the contaminant and other impurities in LMWHs is critically important.

6. Summary and Conclusions HIT is an immune-mediated serious adverse effect of heparin exposure that results in platelet activation, inflammation, and an extreme hypercoagulable state. HIT antibodies cause platelet activation, platelet aggregation, the generation of procoagulant platelet microparticles, and activation of leukocytes and endothelial cells. The clinical manifestations are thrombocytopenia and a high rate of thromboembolic complications that can progress to amputation or death. Early diagnosis based on a comprehensive interpretation of clinical and laboratory information improves clinical outcomes. Careful monitoring for thrombocytopenia and thrombosis during and for at least several days following heparin treatment of any dose and duration is important. Limitations of the laboratory assays and atypical clinical presentations often make the diagnosis of HIT difficult. The ELISA and platelet function laboratory tests for HIT provide different information. The ELISA tests merely provide evidence of the presence of HIT antibodies that may or may not be clinically relevant. Thus, specificity of ELISA tests is low. Platelet function tests detect HIT antibodies that cause platelet activation and have a better correlation to patients with HIT-associated thrombocytopenia and thrombosis. Because of a lower sensitivity, a negative platelet function test result cannot exclude HIT. A combination of tests repeated over several days provides the best information. All heparins, including LMWH, must be stopped when the diagnosis of HIT is suspected. For the management of HIT thrombosis, the DTIs argatroban or lepirudin, or the FXa inhibitor danaparoid, are recommended. Differences between

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drugs need to be considered when making a clinical treatment decision for thrombosis prophylaxis, thrombosis treatment, and interventional procedures. For long-term management of HIT thrombosis VKAs are used, but specific dosing guidelines must be followed. Bivalirudin and fondaparinux may be other potential treatment options but these have not been fully studied in this patient population. In the future, idraparinux or the oral FXa or thrombin inhibitors may prove to be useful if clinically validated. The diagnosis and treatment of HIT is complex, but needs to be considered in the clinical management of patients exposed to heparin due to its serious outcomes. Research and clinical studies will continue to address the unresolved issues and unmet clinical needs associated with HIT. References 1. Kelton, J.G., Sheridan, D., Santos, A., Smith, J., Steeves, K., Smith, C., Brown, C., and Murphy, W.G. (1988) Heparin-induced thrombocytopenia: laboratory studies Blood 72, 925–30. 2. Amiral, J., Bridey, F., Dreyfus, M., Vissac, M., Fressinaud, E., Wolf, M., and Meyer, D. (1992) Platelet factor 4 complexed to heparin is the target for antibodies generated in heparin-induced thrombocytopenia Thromb Haemost 68, 95–6. 3. Greinacher, A., Potzsch, B., Amiral, J., Dummel, V., Eichner, A., and MuellerEckhardt, C. (1994) Heparin-associated thrombocytopenia: isolation of the antibody and characterization of a multimolecular PF4-heparin complex as the major antigen Thromb Haemost 71, 247–51. 4. Maccarana, M., and Lindahl, U. (1993) Model of interaction between platelet factor 4 and heparin Glycobiology 3, 271–7. 5. Mayo, K.H., Ilyina, E., Roongta, V., Dundas, M., Joseph, J., Lai, C.K., Maione, T., and Daly, T.J. (1995) Heparin binding to platelet factor-4. An NMR and site-directed .mutagenesis study: arginine residues are crucial for binding Biochem J 312, 357–65. 6. Newman, P.M., and Chong, B.H. (2000) Heparin-induced thrombocytopenia: new evidence for the dynamic binding of purified anti-PF4-heparin antibodies to platelets and the resultant platelet activation Blood 96, 182–7. 7. Suh, J., Aster, R.H., and Visentin, G.P. (1998) Antibodies from patients with heparin-induced thrombocytopenia/thrombosis recognize different epitopes on heparin:platelet factor 4 Blood 91, 916–22.

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Chapter 5 Novel Anticoagulant Therapy: Principle and Practice Shaker A. Mousa Abstract Currently, there are several lines of evidence supporting the interplay between coagulation and inflammation in the propagation of various disease processes, including venous thromboembolism (VTE) and inflammatory diseases. Major advances in the development of oral anticoagulants have resulted in considerable progress toward the goal of safe and effective oral anticoagulants that do not require frequent monitoring or dose adjustment and have minimal food/drug interactions. Indirect inhibitors such as low-molecular-weight heparin (LMWH) and the pentasaccharide fondaparinux represent improvements over traditional drugs such as unfractionated heparin for acute treatment of VTE, constituting a more targeted anticoagulant approach with predictable pharmacokinetic profiles and no requirement for monitoring. Vitamin K antagonist, with its inherent limitations in terms of multiple food and drug interactions and frequent need for monitoring, remains the only oral anticoagulant approved for long-term secondary thromboprophylaxis in VTE. The oral-direct thrombin inhibitor ximelagatran was withdrawn from the world market due to safety concerns. Newer anticoagulant drugs such as parenteral pentasaccharides (idraparinux, SSR126517E), novel oral-direct thrombin inhibitors (dabigatran), oral-direct factor Xa inhibitors (rivaroxaban, apixaban, YM-150, DU-176b), and tissue factor/factor VIIa complex inhibitors have been “tailor-made” to target specific procoagulant complexes and have the potential to greatly expand oral antithrombotic targets for both acute and long-term treatment of VTE, acute coronary syndromes, and for the prevention of stroke in atrial fibrillation patients. Key words: Anticoagulants, direct thrombin inhibitors, factor Xa inhibitors, factor IXa inhibitors, pentasaccharide, tissue factor/factor VIIa complex inhibitors, venous thromboembolism.

1. Introduction Inflammation plays a key role in triggering a prothrombotic state through the activation of platelets, induction of coagulation, and vascular insult. Venous thromboembolism (VTE) continues to be a major cause of morbidity and mortality in the western world S.A. Mousa (ed.), Anticoagulants, Antiplatelets, and Thrombolytics, Methods in Molecular Biology 663, DOI 10.1007/978-1-60761-803-4_5, © Springer Science+Business Media, LLC 2003, 2010

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(1). VTE represents 1 in 10 hospital deaths, and post-thrombotic syndrome and pulmonary hypertension occur in 10% of deep-vein thrombosis (DVT) and 5% of pulmonary embolism (PE) patients, respectively (2). For more than 50 years, traditional drugs, such as unfractionated heparin (UFH), have been used parenterally for acute treatment, followed by oral vitamin K antagonists (VKAs) such as warfarin for long-term treatment. These drugs exert their antithrombotic effects by inhibiting multiple steps of the coagulation cascade, but there are inherent limitations for each drug. For acute VTE treatment, the limitations of UFH include a less than predictable anticoagulant response with the need for frequent monitoring, and the potential for severe toxicity, especially heparin-induced thrombocytopenia (HIT) in up to 3% of patients (3). Over the past 15 years, the use of LMWH and more recently the synthetically derived pentasaccharide fondaparinux has improved the acute management of VTE. A more targeted approach to procoagulant complex inhibition, predictable pharmacodynamic characteristics, and improved safety profiles have enabled complete treatment of VTE on an outpatient basis for select patients without the need for anticoagulant monitoring. Other parenteral drugs such as the direct thrombin inhibitors (DTIs) lepirudin and argatroban have achieved only limited use in acute VTE treatment, namely in thrombosis associated with HIT. Optimal long-term treatment of VTE is defined by the limitations of VKAs, the only oral anticoagulants currently approved for use in this setting. These limitations include a slow onset of action and the need for bridging anticoagulation with a parenteral drug in the acute setting, multiple food and drug interactions, and a narrow therapeutic window, necessitating frequent coagulation monitoring and dose adjustment (4). In addition, some patient subgroups cannot tolerate VKA, such as pregnant patients requiring anticoagulation, in whom VKA is associated with a risk of teratogenicity (5), or patients in whom VKA is associated with higher risks of recurrent thromboembolism and major bleeding, such as those with active cancer (6, 7). In both of these patient groups, emerging data support the use of long-term LMWH (8– 10), with limited parenteral use. An improved understanding of the molecular mechanisms of coagulation and thrombosis and the potential to apply this knowledge at the clinical level to different patient subgroups has led to the development of newer antithrombotic drugs for use in VTE treatment. Many of these drugs are orally active, synthetically derived, and target-specific procoagulant complexes within the coagulation cascade (11). These drugs can broadly be categorized as interfering with the initiation of coagulation [tissue factor/factor VIIa (TF/FVIIa) complex], propagation of coagulation [indirect and direct inhibitors of activated factor X (FXa)

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or FIXa], and thrombin activity (DTIs). This chapter will focus on these types of investigational drugs for VTE treatment, with an emphasis on those undergoing or that have recently completed phase-II or III clinical trials (Table 5.1). The effect of anticoagulants on stroke in atrial fibrillation patients will also be discussed. Atrial fibrillation (AF) is an epidemic, affecting 11.5% of the population in the developed world. Its significance lies predominantly in that AF patients have a fivefold increased risk of stroke. Strokes associated with AF are usually more severe and confer increased risk of morbidity and mortality and poor functional outcomes. Despite advances in the development of promising experimental approaches for select patients with acute stroke, primary prevention with pharmacological agents remains the best approach to reducing the burden of stroke.

2. Anticoagulant Targets That Inhibit the Initiation Phase 2.1. TF/FVIIa Complex Inhibitors

The TF/FVIIa complex, as part of the extrinsic system of the coagulation cascade, is considered to be the key system for the initiation of coagulation. In the venous vascular system, exposure of TF in orthopedic surgery and in subsets of cancer patients (12, 13) is believed to be responsible for the high risk of VTE in these patient groups, making pharmacological inhibition of the TF/FVIIa complex important (14). The function of TF can be blocked using several approaches, including antibodies that prevent the binding of FVIIa to TF, active site inhibition of FVIIa, small molecules or antibodies that block TF/FVIIa complex function, and molecules that inhibit the active site of FVIIa in the TF/FVIIa complex after binding to FXa (15, 16). Moreover, TF pathway inhibitor (TFPI), a naturally occurring inhibitor, forms a neutralizing complex with TF/FVIIa and FXa (14). Thus TFPI, either by upregulation of endogenous TFPI pools or exogenous administration of recombinant TFPI (rTFPI), represents an attractive anticoagulant that acts by blocking the pathological impact of TF/VIIa and Xa on coagulation and beyond (17).

2.2. Nematode Anticoagulant Proteins

The hematophagous hookworm Ancylostoma caninum produces a family of small, disulfide-linked protein anticoagulants (75–84 amino acid residues) referred to as nematode anticoagulant proteins (NAPs), which have been the focus of antithrombotic drug development efforts due to their ability to inhibit the TF/FVIIa

Factor Xa

Factor Xa

Factor Xa

Factor IIa (thrombin)

Rivaroxaban (Bayer Schering)

Apixaban (Bristol-Myers Squibb and Pfizer)

LY517717 (Eli Lilly),

Dabigatran (Boehringer Ingelheim)

Once or twice daily

Once daily

Twice daily

Once or twice daily

Dosing

• Phase III for VTE prophylaxis after joint replacement completed (n=2,531) • Phase II for secondary prevention after ACS enrolling (n=1,350) • Phase II for treatment of DVT completed, phase-III enrolling (n=2,900) • Phase III for prevention of stroke in non-valvular AF enrolling (n=14,000) • Phase III for treatment of PE enrolling (n=3,300) • Phase II trial for treatment of acute DVT completed • Phase III trials for VTE prevention after joint replacement enrolling (n=7,258) • Phase III trial for prevention of VTE in medical patients (n=6,524) • Phase III trial for prevention of stroke in non-valvular AF enrolling (n=15,000) • Phase II trial for VTE prophylaxis after joint replacement completed • Phase III trial for VTE prevention after joint replacement completed • Phase III trial for treatment of VTE enrolling (n=2,554) • Phase III trial for prevention of stroke in non valvular AF enrolling (n=15,000)

• Avoid or monitor with strong CYP450 3A4 inhibitors

• Undetermined yet • Avoid or monitor with proton pump inhibitors

Indications

• Avoid or monitor with CrCl <30 ml/min • Dose reduction for CrCl 30–49 ml/min • Avoid or monitor with strong CYP450 3A4 inhibitors

Adjustment and interactions

a Additional oral anticoagulants in preclinical or early clinical developments are not listed.

Target

Druga

Table 5.1 Examples of oral anticoagulants in phase-II/III trials

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complex. One of these nematode anticoagulant proteins, NAP5, inhibits the amidolytic activity of FXa with a Ki = 43 pM and is the most potent natural FXa inhibitor identified to date. NAP5 does not inhibit FVIIa in the TF/FVIIa complex. Rather, it either binds to FXa alone, or, as is the case for its family member NAPc2, in combination with a protein exosite, resulting in potent inhibition of the TF/FVIIa complex. NAPc2 has been tested subcutaneously for VTE prophylaxis in a phase I/II clinical trial using mandatory unilateral venography in 293 patients undergoing total knee replacement surgery. At a dose of 3 μg/kg administered within 1 h after surgery, NAPc2 was associated with an overall DVT rate of 12.2%, a proximal DVT rate of 1.3%, and a major bleed rate of 2.3% (18). Further clinical trials with NAPc2 are in progress (19, 20). Recombinant NAPc2, like other inhibitors of TF/FVIIa, including TFPI and active site-blocked FVIIa (ASIS, FFR-rFVIIa, or FVIIai) have a promising role in the prevention and treatment of venous and arterial thrombosis and could potentially be efficacious in the management of disseminated intravascular coagulopathies due to their ability to potentently and selectively inhibit TF/FVIIa (19, 20). 2.3. Anti-TF/VIIa

FVIIa is a key serine protease involved in the initiation of the coagulation cascade. It is a glycosylated disulfide-linked heterodimer comprised of a heavy chain and a light chain. The light chain contains an amino-terminal gamma-carboxyglutamic acid-rich (Gla) domain and two epidermal growth factor (EGF)like domains, and there is a chymotrypsin-like serine protease domain in the heavy chain (21). TF, a membrane bound protein, is an essential cofactor of FVIIa that is required for maximal activity toward its biological substrates (FX, FIX, and FFVII). As such, the TF/FVIIa complex plays an important role in normal physiology as well as in thrombotic diseases such as unstable angina (UA), disseminated intravascular coagulation (DIC), and DVT. In addition to its function as an initiator of coagulation, TF/FVIIa plays an important role in inflammation and angiogenesis (22, 23). A wide array of strategic approaches to inhibiting the biochemical and biological functions of the TF/FVIIa complex have been pursued. These have been greatly enhanced by elucidation of the structures of TF, FVII, FVIIa, and the TF/FVIIa complex, resulting in inhibitors that are directed specifically toward either FVIIa or TF. Antagonists of the TF/FIIa complex include active site-inhibited FVIIa, TF mutants, anti-TF antibodies, antiFVII/FVIIa antibodies, naturally occurring protein inhibitors, peptide exosite inhibitors, and protein and small-molecule active site inhibitors. These antagonists can inhibit catalysis directly at the active site as well as impair function by binding to exosites that interfere with substrate, membrane, or cofactor binding. Several different small-molecule potent inhibitors of TF/FVIIa have

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been shown to reduce thrombus weight in animal models and decrease the level of interleukin-6 (IL-6) in a LPS-stimulated mouse model of endotoxemia (22–25). A study designed to evaluate the antithrombotic efficacy and bleeding propensity of a selective, small-molecule inhibitor of TF/VIIa in comparison to small-molecule, selective inhibitors of FXa and thrombin in a nonhuman primate model of thrombosis was reported by Suleymanov et al. (25). The data indicated that TF/VIIa inhibition effectively prevents arterial thrombosis, with less impact on bleeding parameters than equivalent doses of FXa and thrombin inhibitors (25). 2.4. TFPI

The anticoagulant effects of TFPI in animals (26, 27) and the role of TFPI release in secondary anticoagulant mechanisms of LMWH action in humans (28) have been demonstrated. To date, rTFPI has been tested only in experimental models. TFPI is a natural (i.e., endogenous) inhibitor of TF coagulant and signaling activities. TFPI exerts anti-angiogenic and antimetastatic effects in vitro and in vivo (17, 29). In animal models of experimental metastasis, circulating and tumor cell-associated TFPI significantly reduce tumor cell-induced coagulation activation and lung metastasis (29). Heparins and heparin derivatives, which induce the release of TFPI from the vascular endothelium, also exhibit antitumor effects, and TFPI likely contributes significantly to these effects (17). Recently, a non-anticoagulant LMWH with intact TFPI-releasing capacity was shown to have significant antimetastatic effects in an experimental mouse model (30). Evidence of dual inhibitory functions of TFPI on TF-driven coagulation and signaling strengthen the rationale for considering TFPI a potential anticancer agent (26). TFPI-2, a member of the Kunitz-type serine proteinase inhibitor family, is a structural homologue of TFPI. The expression of TFPI-2 in tumors is inversely related to the degree of malignancy, suggesting a role for TFPI-2 in the maintenance of tumor stability and inhibition of growth of neoplasms (31). TFPI2 inhibits the TF/VIIa complex and a wide variety of serine proteinases, including plasmin, plasma kallikrein, FXIa, trypsin, and chymotrypsin. Aberrant methylation of TFPI-2 promoter cytosine-phosphorothioate-guanine (CpG) islands in human cancers and cancer cell lines results in decreased expression of TFPI-2 mRNA and decreased synthesis of TFPI-2 protein during cancer progression (31). TFPI-2 has been shown to induce apoptosis and inhibit angiogenesis, thereby potentially contributing significantly to inhibition of tumor growth. Restoration of TFPI-2 expression in tumor tissue inhibits invasion, tumor growth, and metastasis, lending support to the use of TFPI-2-targeted therapeutics as novel treatments for cancer (31).

Novel Anticoagulant Therapy: Principle and Practice

3. Inhibitors of Coagulation Propagation

3.1. Indirect FXa Inhibitors

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It is widely accepted that FXa, as part of a prothrombinase complex with FVa, plays a central role in clot formation, given that it is generated by the extrinsic and intrinsic pathways of coagulation as they converge into the final common pathway. In addition, within the prothrombinase complex, one molecule of FXa can exponentially generate 138 molecules of thrombin per minute. Theoretically, FXa inhibitors might have an advantage over thrombin inhibitors by preventing the activation of coagulation amplification mechanisms as well as thrombin generation in both platelet-rich arterial thrombosis and fibrin-rich venous thrombosis, making FXa a prime target for anticoagulant drug design. However, in animal models of thrombosis, selective FXa inhibition was less potent than direct thrombin inhibition in arterial and venous models of thrombosis, and associated with increased global clotting times, indicating that inhibition of FXa is less effective than direct thrombin inhibition in controlling thrombin formation (32). The possibility that selective inhibition of FXa upstream could result in a safer bleeding profile cannot be entirely ruled out; in the absence of thrombin inhibition, small amounts of thrombin would escape neutralization and facilitate hemostasis. Inhibitors of FXa include both indirect (antithrombinmediated) and direct antithrombin (AT)-independent selective inhibitors. Other possible targets of coagulation propagation, through the prothrombinase complex or other routes, include FIXa inhibitors, FVIIIa and FVa inhibitors, activated protein C, or soluble thrombomodulin. The synthetically derived pentasaccharides fondaparinux and idraparinux represent the most advanced selective indirect FXa inhibitors. These agents exert their action through high-affinity binding and activation of AT, which then inhibits free FXa. Fondaparinux contains the pentasaccharide sequence of heparin and selectively binds to and induces a conformational change in AT, increasing the anti-Xa activity of AT nearly 300-fold in a catalytic fashion. Fondaparinux has a linear pharmacokinetic profile and predictable anticoagulant response, with a plasma half-life of approximately 18 h and >95% bioavailability after intravenous or subcutaneous injection, allowing non-monitored once-daily subcutaneous dosing. In addition, it does not bind to platelet factor 4 (PF4) and has not been associated with drug-induced thrombocytopenia. Fondaparinux is currently approved for acute treatment of DVT and PE based on the recently completed MATISSE studies in VTE (33, 34). As such, fondaparinux represents the first

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of a class of selective indirect FXa inhibitors to provide proof of concept that FXa inhibitors can be used to treat thrombosis in the acute stage as efficaciously as drugs with established AT activity. Fondaparinux is also the first of a new class of antithrombotic drugs designed specifically to inhibit a single target or procoagulant complex in the coagulation cascade. 3.1.1. Idraparinux (Sanofi-Aventis)

Idraparinux is a hypermethylated, long-acting pentasaccharide that can be administered with once-weekly dosing. Idraparinux sodium is a second-generation pentasaccharide with sulfated side chains, which results in a 30-fold higher binding affinity to AT as compared to fondaparinux, and a 120-h elimination half-life, allowing once-weekly administration (35). It has similar clinical properties as fondaparinux, with 100% bioavailability after parenteral administration, linear pharmacokinetics, a predictable anticoagulant response with no need for monitoring, does not induce platelet aggregation, has no effects on PF4, and no evidence of induction of thrombocytopenia. The major drawback, as for fondaparinux, has been the lack of an antidote, although the importance of an antidote in clinical practice is controversial. However, it should be noted that biotinylated idraparinux, discussed in detail later, does have an antidote. The PERSIST study, a randomized, phase-II, dose-ranging study, compared idraparinux with warfarin over a 12-week course of treatment for DVT (after initial studies of enoxaparin with idraparinux demonstrated efficacy at all doses that was similar to warfarin). No clear dose–response relationship for efficacy was shown with idraparinux, but a significant dose–response relationship for major bleeding was shown (36). A large phase-III trial comparing the efficacy and safety of idraparinux with heparin or fondaparinux and dose-adjusted warfarin in both acute and long-term treatment of DVT and PE has recently been completed (36).

3.1.2. SSR126517E (Biotinylated Idraparinux)

This synthetic pentasaccharide being developed by Sanofi-Aventis exerts antithrombotic properties through AT-mediated inhibition of FXa activity. It is identical to idraparinux with the exception of a biotin moiety covalently affixed through a linker to the pentasaccharide structure so that anti-FXa activity can be neutralized in vivo by avidin. In vitro studies revealed that SSR126517E binds with high-affinity (Kd = 10.9 mol/l) to human AT and inhibits FXa in a concentration-dependent manner. It does not inhibit platelet aggregation or cross-react with antibodies from sera of patients with HIT. In phase-I studies, the median time to reach maximum concentration was 4 h, with an absolute bioavailability of 100% and a half-life of approximately 200 h. Exposure of SSR126517E to avidin resulted in a rapid decrease of anti-Xa activity and no serious adverse events (37).

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3.2. Selective Direct FXa Inhibitors

Advantages of direct FXa inhibitors include the absence of intermediary molecules such as AT that may potentially contribute to inconsistent anticoagulation, particularly in acute or inflammatory states. DX-9065a was the first of a class of small, synthesized, selective direct FXa inhibitors to undergo phase-II clinical trials in arterial thrombosis (38, 39). Efforts to develop orally available selective FXa inhibitors for VTE and prevention of stroke in AF patients are underway (see Table 5.1).

3.2.1. Razaxaban

Razaxaban (BMS-561389), developed by Bristol-Myers Squibb (formerly Dupont), represents the first of a new class of synthetically derived, small-molecule, oral-direct FXa inhibitors that do not require anticoagulant monitoring. In phase-I trials involving young and elderly volunteers, it was well tolerated and well absorbed with only nuisance bleeding reported; FXa inhibition and dose-dependent anticoagulation were noted. Razaxaban was investigated for proof of principle in DVT prevention in patients undergoing total knee replacement. In phase-IIb trials, razaxaban at doses of 25, 50, 75, or 100 mg twice daily administered starting 8 h after surgery was compared to enoxaparin at a dose of 30 mg twice daily initiated 12–24 h after surgery. The study revealed efficacy but an unacceptable risk/benefit profile at higher doses (40). Razaxaban was discontinued for further development in March 2005 in lieu of an oral FXa inhibitor under development by the same company with a more favorable safety profile (see below).

3.2.2. Apixaban

Apixaban (formerly BMS-562247 or DPC-AG0023) is an orally active, small-molecule direct FXa inhibitor being developed by Bristol-Myers Squibb that has a more favorable safety profile than razaxaban. It is a highly potent inhibitor of human FXa, with a Ki of 0.08 ± 0.01 nM, and binds to serum proteins at a rate of 87%. It has a consistent oral absorption profile and linear pharmacokinetics, with a maximal plasma concentration achieved within 3 h and an effective half-life of 9 h for twice-daily administration, and 14 h for once-daily administration. It is eliminated via both hepatic and renal routes. Apixaban had only modest effects on two traditional markers of anticoagulation, international normalized ratio (INR) and activated partial thromboplastin time (aPTT). Phase-I clinical studies revealed mild bleeding and prolonged bleeding time, with no evidence of elevated transaminases (alanine transaminase or aspartate transaminase ≥ 5 times the upper limit of the normal range). The safety profile of apixaban remains to be determined in phase-II/III trials, and it is presently undergoing phase-II clinical studies in elective total knee replacement surgery patients and patients with acute DVT.

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The efficacy and safety of apixaban as a thromboprophylaxis in patients following total knee replacement was determined (41). Apixaban in doses of 2.5 mg twice daily or 5 mg once daily exhibited a promising benefit-risk profile as compared to current standards of care following total knee replacement (41). 3.2.3. Rivaroxaban (Bayer HealthCare AG and J&J/Scios, Inc.)

Rivaroxaban (formerly BAY 59-7939) is a small-molecule, selective oral-direct FXa inhibitor developed by Bayer for the prevention and treatment of thrombosis. Oral rivaroxaban may be given in fixed once-daily doses, with potentially no coagulation monitoring. These properties, along with results from preclinical and clinical studies, suggest that the use of rivaroxaban might be more advantageous than current treatments. Studies in arterial and venous animal models have demonstrated that rivaroxaban has potent antithrombotic properties without prolonging bleeding times. In healthy subjects, rivaroxaban was well tolerated with a predictable pharmacological profile and a low propensity for clinically relevant drug–drug interactions. In preclinical studies, endogenously generated FXa was inhibited with an IC50 of 21 ± 1 nM (42). This antithrombotic effect was demonstrated in different thrombosis models at doses of 0.6–10 mg/kg, depending on the model and species, following oral administration. In dogs, bioavailability ranged from 60–86%. In phase-I studies, rivaroxaban was rapidly absorbed [maximum concentration (Cmax) achieved after 30 min] and well tolerated (up to 80 mg single dose in healthy people) (43). Elimination occurred with a terminal half-life of 4.86–9.15 h (steady state). Prothrombin time (PT), aPTT, and HepTest were prolonged in a dose-dependent manner, and there was no effect on bleeding time. In elderly men and women (>60 years), mean area under the concentration–time curve and Cmax tended to be about 20% higher. There were no drug–drug interactions or induction of major cytochrome P450 (CYP) isoforms, with the exception of strong CYP3A4 inhibitors, and no prolongation of QTc was observed. Four large dose-ranging studies of rivaroxaban (ODIXa-HIP, ODIXa-HIP2, ODIXa-KNEE, and ODIXa-OD-HIP) have been completed, covering a 12-fold increase in dose, from 2.5 to 30 mg twice daily and 5 to 40 mg once daily for VTE prevention following major orthopedic surgery (44, 45). The open-label phaseIIa ODIXa-HIP trial using mandatory venography confirmed proof of principle for rivaroxaban. Other studies have confirmed the efficacy and safety of rivaroxaban as compared to enoxaparin under double-blind, double-dummy conditions for VTE prevention in patients undergoing orthopedic surgery (44). Phase-II studies of rivaroxaban for the prevention of VTE after major orthopedic surgery support these findings and suggest that a total

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daily dose range of 5–20 mg rivaroxaban has similar efficacy and safety as enoxaparin, with 10 mg rivaroxaban once daily the optimal dose (46). The drug development plan for rivaroxaban is aggressive, with a program of simultaneous investigation of multiple indications rather than a sequential approach. At present, over 24,000 patients (12,000 are predominantly post-operative orthopedic patients) have been evaluated in completed phase-II and III trials of rivaroxaban for thromboprophylaxis and treatment of DVT. By the time all the currently enrolling trials have concluded, more than 50,000 patients will have been evaluated in randomized controlled trials of rivaroxaban. The advantages of rivaroxaban include the potential for once-daily dosing for all indications, no required dose adjustment for body weight, no known interactions with common cardiovascular medications, a relatively safe pharmacodynamic profile with respect to bleeding risk and hepatotoxicity, no clinically significant interaction with aspirin, and the ability to bridge with LMWH when necessary. On the other hand, rivaroxaban is partially renally cleared and will require dose adjustment in those with grade-III chronic kidney disease and is not being studied in patients with a creatinine clearance less than 30 ml/min. In addition, since rivaroxaban metabolism is affected by potent cytochrome P450 3A4 inhibitors such as ketoconazole and clarithromycin, and protease inhibitors, use will be restricted in certain special populations. Nonetheless, after extensive phase-II, and now emerging phase-III trial data, it appears that rivaroxaban is effective in preventing and treating VTE with a bleeding risk comparable to other anticoagulants. The results of randomized trials evaluating rivaroxaban for the prevention of stroke and non-central nervous system (CNS) embolism in AF and secondary prevention of acute coronary syndromes are currently ongoing. While there are several other oral anticoagulants in development, none have been evaluated as extensively or in as many patients as rivaroxaban. Rivaroxaban has the advantage that it can be dosed once a day, which has been shown to improve patient compliance and outcomes (47, 48). Despite once-daily dosing, rivaroxaban has a half-life that is considerably shorter than other oral FXa inhibitors, which is advantageous in the event of bleeding or an urgent need to discontinue anticoagulation. Unlike the DTI dabigatran, the bioavailability of rivaroxaban is very good, and there is low risk of drug–drug interactions, including with medications that alter gastric pH, which are taken chronically by 3% of the US population (49). Perhaps more importantly, rivaroxaban has been shown to have no effect on platelet aggregation, and its pharmacokinetic profile is unaffected by aspirin and other notable cardiovascular medications such as digoxin. Finally, after clinical investigation in thousands of patients, rivaroxaban appears

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to have no significant hepatotoxicity and a bleeding risk comparable to other conventional anticoagulants. Rivaroxaban could potentially be used in several clinical indications and disease states, including VTE prophylaxis, long-term treatment of DVT, PE, and the prevention of stroke and nonCNS embolism in patients with AF and possibly acute coronary syndromes. In order to understand the efficacy and safety of a therapeutic across such a wide range of patient populations, several large simultaneous studies evaluating rivaroxaban in both venous and arterial thromboembolism are under way. The greatest therapeutic impact of rivaroxaban might be in providing a much-needed and attractive alternative to warfarin. While formal cost-effective analyses are not yet available, avoiding the intense, costly, and frequent monitoring required with VKAs, as well as the potential to reduce adverse vascular events precipitated by the narrow therapeutic window of VKAs, will most likely translate into a significant improvement in quality of life and cost savings. While further data (especially large phase-III trials) and caution are required, there is reason for optimism. Rivaroxaban may very well be the long-awaited alternative to warfarin. 3.2.4. YM-150

Astellas (formerly Yamanouchi) has developed YM-150, an oralselective FXa inhibitor for DVT prevention. The compound has an immediate antithrombotic effect after oral administration, with a dose-dependent response and prolongation of PT; no significant food interactions have been noted. In a phaseII dose-escalation study in patients undergoing elective primary hip replacement surgery, YM-150 (3, 10, 30, or 60 mg PO once daily) given 6–10 h after surgery for 7–10 days was compared to enoxaparin administered at a dose of 40 mg SQ once daily 12 h before surgery (50). There was no major bleeding, and the median incidence of VTE ranged from 52% in the 3-mg group to 19% in the 60-mg group. Overall, the drug appears to be safe and well tolerated. A dose-escalation study of YM150 in the prevention of VTE in elective primary hip replacement surgery was also carried out (51). YM150, administered at doses of 10–60 mg daily starting 6–10 h after primary hip replacement was shown to be safe, well tolerated, and effective.

3.2.5. DU-176b

Daiichi Sankyo is developing DU-176b, an oral FXa inhibitor for the treatment of thrombotic disorders. Preclinical data in mouse models revealed potent antithrombotic effects in AT-positive and AT-deficient mice (52). In rat models, DU-176b at doses of 0.05– 1.25 mg/kg/h prevented arterial and venous thrombosis. In terms of clinical studies, DU-1766 in a single 60-mg dose was given to healthy males. The drug inhibited FXa activity, reduced thrombin generation, prolonged PT, aPTT and INR, and

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reduced venous and arterial thrombosis by 28 and 26%, respectively, in a Badimon chamber. Further studies are planned. 3.2.6. LY-517717

LY-517717, an indol-6-yl-carbonyl derivative, is the lead in a series of oral-selective FXa inhibitors being developed by Eli Lilly as part of a research collaboration with Protherics for the potential treatment of thromboembolic diseases. It is 1000-fold more selective as an FXa inhibitor than other serine proteases, with a Ki of 5 nM. The oral bioavailability of LY-517717 is approximately 25–82%, with a plasma half-life of 7–10 h. In a rat atrioventricular shunt model, the median effective dose was 5–10 mg/kg PO, and absorption in dogs indicated no bleeding issues. In a phase-I study, LY-517717 was found to be well tolerated and suitable for once-daily administration. In a dose-escalating study, 511 patients undergoing hip or knee replacement surgery were randomized to receive one of the six oral doses of LY-517717 (25, 50, 75, 100, 125, or 150 mg), or enoxaparin (40 mg SQ daily), started preoperatively, for 6–10 doses. The 100, 125, and 150-mg dose groups were non-inferior to enoxaparin in the incidence of symptomatic or venographically proven DVT or PE (53). The compound produced a dose-dependent prolongation of PT and was well tolerated, and there were no differences in bleeding risk as compared to enoxaparin (53).

3.3. Selective, Direct FIXa and FXI Inhibitors

Although the development of direct FIXa inhibitors is at an earlier phase than direct FXa inhibitors, the theoretical advantages are similar. TTP 889, manufactured by Trans Tech Pharma, is an oral, direct FIXa inhibitor with a half-life of 20 h, enabling once-daily dosing. A phase-II proof of principle study for VTE prevention in hip fracture surgery, the FIXIT trial, recently completed enrollment of 206 patients who received standard in-hospital thromboprophylaxis. Efficacy and safety are being compared between patients randomized to receive TTP 889 versus placebo for up to 3 weeks post-discharge (54). Development of FXIa inhibitors is currently at the preclinical level. BMS-262084 is an irreversible and selective small-molecule inhibitor of FXIa, with an IC50 of 2.8 nM against human FXIa. The effect of inhibiting activated blood coagulation FXIa with BMS-262084 has been determined in rat models of thrombosis and hemostasis (55). BMS-262084 doubled aPTT in human and rat plasma at concentrations of 0.14 and 2.2 μM, respectively. Consistent with FXIa inhibition, PT was unaffected at concentrations up to 100 μM. BMS-262084 administered by intravenous loading with sustained infusion was effective against FeCl(2)induced thrombosis in both the vena cava and the carotid artery. In contrast, doses of up to 24 mg/kg +24 mg/kg/h had no effect on TF-induced venous thrombosis or ex vivo PT. Doses of up to 24 mg/kg+24 mg/kg/h did not significantly prolong

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bleeding time provoked by puncture of small mesenteric blood vessels, template incision of the renal cortex, or cuticle incision. These results demonstrate that pharmacologic inhibition of FXIa achieves antithrombotic efficacy with minimal effects on provoked bleeding (55).

4. Inhibitors of Thrombin Activity Thrombin is the central serine protease in hemostasis. The mechanisms of action of thrombin involve coagulation, platelet activation, fibrinolysis, and vascular cell biology. In addition to its major role in fibrin formation and activation of FXIII, which cross-links fibrin, thrombin is essential for feedback activation of other coagulation factors such as FV, FVIII, and FIX (56). Thrombin is involved in platelet activation and subsequent aggregation (53) and can act as an anticoagulant by binding to thrombomodulin, which converts protein C to its active form, inactivating FVa and FVIIIa. Thrombomodulin-bound thrombin regulates coagulation through activation of thrombin-activatable fibrinolysis inhibitor (TAFI) and subsequent down-regulation of fibrinolysis. Predictions that thrombin inhibitors would be more effective than FXa inhibitors in arterial thromboembolic disease (where thrombin has a key role in platelet activation) and less effective in VTE have not been borne out by clinical data. Given its central role in the coagulation cascade, inhibitors of thrombin activity – whether mediated by AT or acting directly on the active site – represent an important class of anticoagulant drugs in our armamentarium. 4.1. Indirect Thrombin Inhibitors

SNAC/Heparin. SNAC (sodium N-[8(2-hydroxybenzoyl) amino]caprylate), developed by Emisphere Technologies, enables macromolecule delivery of the large negatively charged and poorly absorbed heparin molecule through a noncovalent complex with heparin, allowing passive, transcellular absorption. SNAC itself has no pharmacological activity. Phase-I studies revealed that in doses of up to 10.5 g SNAC/150,000 U heparin, the compound is well tolerated, with nausea being the only significant adverse event observed. Dose-dependent increases in aPTT and anti-FXa levels were also observed, suggesting that both AT-mediated thrombin and FXa inhibition play a role in the anticoagulant effects of the drug (57). In addition, there was an apparent food and diurnal effect, but no age effect. The PROTECT study was a large phase-III study in 2,264 hip replacement patients with two SNAC treatment arms (low-dose SNAC and high-dose SNAC) for 30 days versus 10 days of enoxaparin at a dose of 30 mg SC every 12 h (58). Mandatory

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venography on days 27–30 revealed an overall VTE rate of 31.8% in the low-dose SNAC group, 29.7% in the high-dose group, and 26.1% in the enoxaparin group. The rates of proximal DVT/PE were 18.6% in the low-dose group and 13.8% in the high-dose group, both of which were significantly higher than in the enoxaparin group (12.7%, P = 0.013 and P = 0.045, respectively). There was overall poor compliance in 22.1% of the patients on the low-dose regimen and in 31.4% of the patients on the high-dose regimen, suggesting that substance compliance was a key factor in failure to achieve proof of principle for the use of SNAC/heparin in VTE prevention as an indirect FIIa/Xa inhibitor. Improved formulations of heparin in solid dosage form are currently under clinical investigation (59, 60). An orally administrable chemical conjugate of heparin and hydrophobic deoxycholic acid, referred to as LHD, has also been developed. LHD was pre-formulated with dimethyl sulfoxide as solubilizer to further improve oral bioavailability (9.1% in monkeys). LHD was absorbed mainly in the jejunum and ileum of the small intestine, although it is in the ileum that absorption was most notable. Mechanistic studies of LHD absorption using Caco-2 cell monolayers, which mimic the intestine, demonstrated that the high permeability of LHD is mediated by passive diffusion through the transcellular route, and permeation is affected in part by bile acid transporters. These results demonstrated the feasibility of using chemically modified heparin for long-term oral administration as an effective therapy for VTE (59). A more recent clinical study determined the true pharmacokinetics for injectable versus oral heparin (60). 4.2. Direct Thrombin Inhibitors

The development of DTIs was driven by three major factors: increasing recognition of immune thrombocytopenia as a potentially severe complication of heparin use (61), the notion that heparin-AT inhibition of thrombin produces only weak inhibition of cell surface- or clot-bound thrombin, which is active and can be released during fibrinolysis (62), and the non-specific binding properties of heparin, necessitating frequent monitoring. Hence, non-AT-based thrombin inhibitors with improved safety profiles over heparin, the ability to inhibit surface- or clot-bound thrombin, and predictable dose responses would be advantageous in the clinical setting. Furthermore, oral formulations of these drugs would be a major advantage. DTIs could be ideal drugs for the treatment of HIT, as this condition is characterized by the generation of large amounts of thrombin. A theoretical concern about DTIs is that they could inhibit the anticoagulant properties of thrombin, namely inhibition of the thrombin–thrombomodulin-mediated negative feedback mechanism of the protein C system, with the possibility of rebound hypercoagulability (63).

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Four parenteral DTIs have emerged: lepirudin, bivalirudin, argatroban, and melagatran, the first three of which have been approved for clinical use. Lepirudin is a naturally occurring bivalent DTI indicated for thromboprophylaxis of HIT. Argatroban is a prototype noncovalent, reversible, small-molecule DTI indicated for thromboprophylaxis or treatment of HIT. Melagatran is the active form of the oral, prodrug, small-molecule DTI ximelagatran, discussed below. All of these drugs have limitations in terms of parenteral use, limited indications, need for frequent monitoring, and high cost. 4.3. Selective Oral-Direct Thrombin Inhibitors 4.3.1. Ximelagatran

Ximelagatran, developed by AstraZeneca, represents the first of a new class of orally active, small-molecule DTIs to reach late-stage development with limited clinical indications for VTE prevention. Ximelagatran is a hydrophilic prodrug that is converted by a cytochrome-P450-independent liver enzyme system to its active form melagatran. Bioavailability of ximelagatran is approximately 20%, and the half-life is 4–5 h in patients. It can be administered twice daily and does not require anticoagulant monitoring or dose adjustment. Ximelagatran was studied extensively in a large phaseIII trial for VTE prevention and treatment and was found to be either superior or equivalent to warfarin in terms of efficacy (64–69). However, initial long-term data with ximelagatran revealed elevated liver enzymes (approximately 6%). Based upon this and other considerations, it was not approved by the US FDA. It was, however, approved in other countries for shortterm, post-orthopedic thromboprophylaxis. In February 2006, AstraZeneca withdrew ximelagatran from the world market due to continuing concerns about severe liver toxicity associated with long-term use.

4.3.2. Dabigatran

Dabigatran etexilate is a small-molecule, orally active, prodrug DTI developed by Boehringer Ingelheim that has reached latestage clinical development. It is rapidly absorbed and converted to the active form, dabigatran. It has linear characteristics in terms of concentration and global coagulation parameters, including thrombin clotting time, INR, and ecarin clotting time. Dabigatran has a Ki of 4.5 ± 0.2 nmol/l, peak plasma concentration 2 h post-dose, and a half-life of approximately 14–17 h after multiple dose administration (70). It is metabolized mainly (80–85%) by renal excretion. The BISTRO II study was a multicenter, parallel group, double-blind, dose-finding study for VTE prevention in 1,949 patients undergoing total hip or knee replacement (71). Patients were randomized to receive dabigatran (50 mg, 150 mg, or

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225 mg twice daily, or 300 mg once daily) starting 14 h after surgery. The comparator was enoxaparin (40 mg once daily) initiated 12 h prior to surgery. A significant dose-dependent decrease in DVT was observed with increasing doses of dabigatran (P < 0.001). Compared to enoxaparin, DVT was significantly lower in patients receiving dabigatran 150 mg twice daily [odds ratio (OR) 0.47, P = 0.0007], 300 mg once daily (OR 0.61, P = 0.02), and 225 mg twice daily (OR 0.47, P = 0.0007). Major bleeding was lower with the low dose of dabigatran (0.3% vs. 2.0%, P = 0.047), but elevated at higher doses, with trends almost reaching statistical significance in those receiving the 300 mg dabigatran dose (4.7%, P = 0.051). In terms of adverse events, the incidence of elevated alanine aminotransferase (>3 times ULN) was lower in the dabigatran groups (1.5–3.1%) than in the enoxaparin group (7.4%). There were no cases of clinically relevant thrombocytopenia. The authors concluded that dabigatran started in the early post-operative period was effective and safe across a wide range of doses. In addition, the frequency and extent of severe hepatic abnormalities were lower than those observed with ximelagatran. Dabigatran is currently undergoing extensive phase-III evaluation for VTE prevention, treatment, and secondary thromboprophylaxis through the RE-VOLUTION program. A randomized, double-blind, non-inferiority trial was conducted comparing dabigatran etexilate to enoxaparin for prevention of VTE after total hip replacement (72). Patients (3,494 total) undergoing total hip replacement were randomized into treatment for 28–35 days with dabigatran etexilate 220 mg (n=1157) or 150 mg (n=1174) once daily, starting with a halfdose 1–4 h after surgery, or SC enoxaparin (40 mg) once daily (n=1162), starting the evening before surgery. The primary efficacy outcome was the composite of total VTE (venographic or symptomatic) and death from all causes during treatment. On the basis of the absolute difference in rates of VTE with enoxaparin versus placebo, the non-inferiority margin for the difference in rates of thromboembolism was defined as 7.7%. Both doses of dabigatran were non-inferior as compared to enoxaparin. There was no significant difference in major bleeding rates with either dose of dabigatran etexilate as compared with enoxaparin (72). The frequency of increased liver enzyme concentrations and acute coronary events during the study did not differ significantly between the groups. The study concluded that oral dabigatran etexilate was as effective as enoxaparin in reducing the risk of VTE after total hip replacement surgery, with a similar safety profile (72). 4.3.3. TGN-167

TGN-167 (TRI-50c-04) is an oral thrombin inhibitor being developed by Trigen Holdings for the potential treatment of

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thrombosis. A controlled release formulation of the drug is also being developed with Eurand for long-term treatment of thrombosis. The compound produces a marked increase in thrombin clotting time, with minimal effects on aPTT. A double-blind, phase-I, dose-escalation study with 20 volunteers showed the drug to be well tolerated, with no significant adverse events reported (73). At 600 mg, all dosed subjects achieved effective anticoagulant activity in vitro. Trigen is planning to continue TGN-167 into phase-II studies.

5. Conclusions The antithrombotic management of VTE will undergo significant changes in the next 5-10 years. Limitations of existing parenteral and oral anticoagulants has led to the development of new agents designed to target specific procoagulant complexes in the coagulation pathway, inhibiting coagulation initiation, coagulation propagation, or thrombin activity. With respect to efficacy, during acute treatment of VTE, newer antithrombotic agents must exhibit at least non-inferiority in a methodologically sound study as compared to the existing parenteral agent of choice, LMWH, and the emerging agent fondaparinux. This is true particularly in high-risk venous thromboses, such as ileofemoral VTE, PE, or VTE associated with cancer. For long-term VTE treatment, there is a need to improve upon existing oral anticoagulants, namely, VKAs. Target-selective oral agents must exhibit an improved safety profile (especially as it pertains to major or clinically significant bleeding), ease of use, and tolerability as compared to VKAs. If successful, emerging oral anticoagulants could negate the traditional distinction of acute versus long-term treatment of VTE, as they could potentially be used throughout the spectrum of disease without the need for overlap with parenteral therapies (52). Lastly, any new long-term anticoagulant must be safely tolerated in combination with antiplatelet agents, as an increasingly aging population will be prone to arterial as well as venous thromboembolic disease. Cost considerations are also important, especially from a populational perspective. Newer agents should, in theory, fulfill the following requirements of an ideal anticoagulant: a rapid onset with predictable response characteristics, predictable pharmacokinetics, pharmacodynamics with low plasma protein binding, no required monitoring, a half-life that provides both safety and ease of use (particularly during temporary withdrawal), lack of food or drug interactions, an excellent safety profile (particularly with respect to immune-mediated thrombocytopenia, hepatotoxicity, and

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potential for thrombotic rebound phenomenon), and reversibility or availability of an antidote. In addition, oral agents with predictable intestinal absorption/bioavailability used in a simple, fixed-dose once or twice daily regimen and for which compliance can be monitored would be even more advantageous. At this time, drugs at the most advanced stage of development with respect to VTE management include the parenteral indirect FXa inhibitor idraparinux and biotinylated idraparinux, the oral DTI dabigatran, and the oral selective direct FXa inhibitors rivaroxaban and apixaban. Whether there are inherent advantages in blocking initial thrombin formation via the prothrombinase complex early in the coagulation system or blocking thrombin directly and preventing feedback amplification is still a matter of debate, as is the notion of whether there is any clinically meaningful effect of small-molecule DTIs that target both clot-bound and free thrombin. Long-term clinical data with respect to efficacy of anti-Xa inhibitors will be available shortly, while long-term data are currently available on the efficacy of direct thrombin inhibition. The lessons from ximelagatran reveal the importance of long-term safety data in different patient populations. Ximelagatran had shown significant potential as a possible replacement to warfarin therapy, but was withdrawn because of potential liver toxicity. In contrast, dabigatran appears to have a better safety profile and has recently entered a phase-III randomized clinical trial for AF. Oral direct FXa inhibitors (rivaroxaban, apixaban, and others) may prove to be more potent and safe. Selective inhibitors of specific coagulation factors involved in the initiation and propagation of the coagulation cascade (FIXa, FVIIa, circulating TF) are at an early stage of development. Additional new agents in clinical development include NAPc2, protein C derivatives, and soluble thrombomodulin.

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inhibitor ximelagatran N Engl J Med 349, 1713–21. Stangier, J., Rathgen, K., Gansser, D., Kohlbrenner, V., and Stassen, J. (2001) Pharmacokinetics of BIBR 953 ZW, a novel low molecular weight direct thrombin inhibitor in healthy volunteers. Abstract Thromb Haemost 86, OC2347. Eriksson, B.I., Dahl, O.E., Buller, H.R., Hettiarachchi, R., Rosencher, N., Bravo, M.L., Ahnfelt, L., Piovella, F., Stangier, J., Kalebo, P., and Reilly, P. (2005) A new oral direct thrombin inhibitor, dabigatran etexilate, compared with enoxaparin for prevention of thromboembolic events following total hip or knee replacement: the BISTRO II randomized trial J Thromb Haemost 3, 103–11. Eriksson, B.I., Dahl, O.E., Rosencher, N., Kurth, A.A., van Dijk, C.N., Frostick, S.P., Prins, M.H., Hettiarachchi, R., Hantel, S., Schnee, J., and Buller, H.R. (2007) Dabigatran etexilate versus enoxaparin for prevention of venous thromboembolism after total hip replacement: a randomised, doubleblind, non-inferiority trial Lancet 370, 949–56. Coombe, S., Allen, G., and Kennedy, A. (2005) A phase I double-blind, ascending dose study of an oral synthetic direct thrombin inhibitor, TGN167 Blood 106, 530a.

Chapter 6 Oral Direct Factor Xa Inhibitors, with Special Emphasis on Rivaroxaban Shaker A. Mousa Abstract Rivaroxaban is a small-molecule, direct factor Xa inhibitor that is under investigation for the prevention and treatment of venous and arterial thrombosis. To date, oral anticoagulants have been limited largely to vitamin K antagonists. Despite their remarkable benefits, vitamin K antagonists are limited by their narrow therapeutic window, the existence of multiple food and drug interactions, and the need for frequent monitoring and dose-adjustment. Rivaroxaban represents a potentially attractive alternative to warfarin, as it could enable simplified once-daily dosing, requires no therapeutic monitoring, and has a lower potential for drug interactions. At present, the safety and efficacy of rivaroxaban for the prophylaxis and treatment of venous thromboembolism has been evaluated in phase-II and phase-III trials involving over 24,000 patients. Rivaroxaban is also being evaluated for the treatment of pulmonary embolism, secondary prevention after acute coronary syndromes, and the prevention of stroke and non-central nervous system embolism in patients with non-valvular atrial fibrillation. The need for new oral anticoagulants, the development and pharmacology of rivaroxaban, results of completed studies of rivaroxaban, and details of ongoing phase-II and phase-III trials with rivaroxaban are the subjects of this chapter. Key words: Oral anticoagulant, rivaroxaban, BAY 59-7939, factor Xa inhibitor, venous thromboembolism.

1. Introduction Arterial thrombosis, venous thrombosis, and subsequent thromboembolism account for significant morbidity and mortality worldwide. In the United States, more than 200,000 patients develop venous thromboembolism (VTE) every year, and 30% of these patients die within 30 days (1). Despite administration of current prophylaxis, 5–20% of all hip replacement surgeries S.A. Mousa (ed.), Anticoagulants, Antiplatelets, and Thrombolytics, Methods in Molecular Biology 663, DOI 10.1007/978-1-60761-803-4_6, © Springer Science+Business Media, LLC 2003, 2010

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are complicated by VTE (2). Furthermore, deep venous thrombosis and pulmonary embolism are associated with a significant economic burden due to the costs of acute care as well as longterm costs associated with recurrent VTE and post-thrombotic syndrome (3). A problem growing at a faster rate is the increasing burden imposed by atrial fibrillation(AF)-associated thromboembolism. AF leads to stasis of blood in the atria and formation of thrombi that can leave the heart and embolize any vascular bed, most seriously the cerebral circulation, leading to stroke. At present, 2.3 million Americans have AF, including 10% of all patients 80 years and older. By 2050, an estimated 5.6 million Americans will have AF, increasing the risk of stroke fivefold, and ultimately accounting for 15–20% of all strokes in the United States (4, 5). Furthermore, patients who have an AF-associated stroke are twice as likely to remain bedridden as other stroke victims (6). While there are several efficacious intravenous and subcutaneous alternatives for acute anticoagulation with a favorable balance of effectiveness and safety in the setting of acute coronary syndromes, deep venous thrombosis, or pulmonary embolism, there are few medications available for chronic, oral anticoagulation in AF. Oral anticoagulation thus far has been limited to vitamin K antagonists (VKAs), principally warfarin sodium. 1.1. Pharmacology of Warfarin

Warfarin, first described by Karl Paul Link in 1940, is the current treatment of choice for the prevention of thromboembolism in patients with AF (7, 8). By interfering with the cyclic interconversion of vitamin K and vitamin K 2,3-epoxide, warfarin impairs the γ-carboxylation of vitamin K-dependent proteins, including important serine proteases in the coagulation cascade that require vitamin K for their biologic activity (Factors II, VII, IX, and X). Warfarin is highly effective in preventing thromboembolic events in patients with AF. In 29 randomized trials involving more than 28,000 patients, warfarin reduced the risk of stroke by 64% (8). Furthermore, warfarin was associated with a 26% (95% CI, 3– 43%) reduction in all-cause mortality in randomized controlled trials when compared to no anticoagulation therapy in patients with AF (8). However, the benefits of warfarin in patients well controlled with the agent might overestimate benefits compared to the effects seen in warfarin-naive patients (9). Despite its effectiveness in preventing stroke and non-central nervous system embolism, warfarin has significant limitations. It has a slow onset of action, narrow therapeutic window, and requires frequent monitoring due to marked inter-individual variation in drug metabolism, as well as multiple drug and food interactions. In fact, patients taking warfarin spend nearly a third of the time outside their target INR window (10). Unfortunately, patients who spend more than 10% of their time outside of their target INR window are more likely to suffer an ischemic stroke and

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mortality (10). Patients with the highest risk of stroke, i.e., elderly individuals with evidence of heart failure, hypertension, diabetes, or prior stroke, have the highest rates of major hemorrhage, with a discontinuation rate up to 26% in the first year of treatment (11). This is likely due to difficulty in maintaining patients within a therapeutic window. Warfarin is a racemic compound, in which the S-warfarin enantiomer is a fivefold more potent vitamin K antagonist than the R-enantiomer (12). Each enantiomer exhibits unique interactions depending on an individual’s genetic background and concomitant medications. Polymorphisms in cytochrome P450 C29 (the enzyme responsible for metabolism of the S-isomer) have been associated with impaired hydroxylation of S-warfarin, leading to low-warfarin dose requirements (13). S-warfarin clearance is preferentially impaired by metronidazole and trimethoprim– sulfamethoxazole, while other medications such as amiodarone inhibit both R- and S-warfarin clearance (14). As a result, predicting the anticoagulation response with drug interactions is extremely complex. Warfarin dosing is further complicated by dramatic interindividual variability in warfarin metabolism. While the mean daily dose is 4.57 mg, more than 5% of patients require a daily dose greater than 10 mg (15). Polymorphisms in the vitamin K receptor gene (VKORC1) have been associated with elevated warfarin dose requirements (16). In addition to difficulties with dosage and frequent and expensive monitoring, warfarin is also associated with significant adverse effects. Long-term warfarin use is associated with a 25% increased risk of osteoporotic fractures (17). Patients on warfarin are at increased risk of life-threatening bleeding, including intracranial hemorrhage, which affected 1.8% of patients older than 75 in the Stroke Prevention in Atrial Fibrillation II (SPAF II) study (18). It has also been observed that the risk of bleeding is highest in the first year of warfarin therapy (9). Finally, as with most therapeutics, there is the following risk/benefit paradox with respect to warfarin-associated bleeding: patients at highest risk of stroke, according to both age and comorbidities, are also the patients with the highest risk of major, life-threatening hemorrhage (11, 19). 1.2. Investigating Potential Alternatives to Warfarin

Due to the limitations and risks associated with warfarin, less than half of eligible patients are ultimately treated with warfarin for stroke prophylaxis (20, 21). Despite these limitations, warfarin has remained the therapy of choice for prevention of thromboembolism in patients with AF since it became clinically available in 1954. Recently, randomized trials have investigated alternatives to warfarin therapy. The Stroke Prevention using an Oral Thrombin Inhibitor in Atrial Fibrillation (SPORTIF) trials compared the use of ximelagatran, a competitive inhibitor of human α-thrombin,

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with dose-adjusted warfarin (22, 23). At the time, ximelagatran represented a favorable alternative to warfarin as it had a predictable anticoagulant profile with rapid oral absorption and a rapid onset of action. Despite promising early results, a review of 6,948 patients treated with ximelagatran revealed transient elevations in alanine aminotransferase greater than 3 times the normal upper limit (ALT > 3×ULN) in 7.9% of patients treated with ximelagatran versus 1.2% in the comparator group (24). In addition to three cases of fatal hepatotoxicity, the clinical trial results also showed a troubling trend toward increased nonfatal myocardial infarction in patients treated with ximelagatran, especially in those in whom it was recently discontinued. As a result, the US Food and Drug Administration did not approve ximelagatran for the prevention of stroke and non-central nervous system (CNS) embolism in patients with AF (25). It was allowed on the market in Europe, but was rapidly pulled by the manufacturer when a fatal case of hepatic failure was attributed to the drug. While warfarin reduces the risk of stroke by 45% (95% CI, 29–57%) compared to antiplatelet monotherapy with aspirin, it is also associated with a 70% increased risk of bleeding (26). The ACTIVE W study was a randomized trial designed to determine whether dual antiplatelet therapy with aspirin and the thienophyridine clopidogrel was non-inferior to adjusted dose warfarin therapy (9). The trial was terminated early due to clear superiority of oral anticoagulation with warfarin. 1.3. Safer and More Efficacious Anticoagulation

The ideal oral anticoagulant would have a wide therapeutic window, rapid onset of action, minimal food and drug interactions, a short half-life allowing for quick termination in the event of bleeding, a readily available antidote or reversal agent, and clear efficacy in large trials without adverse effects. When considering candidates for potential new oral anticoagulants, attention must be paid to the three temporal aspects of hemostasis, including (1) initiation, (2) amplification, and (3) termination (27). The prototypical anticoagulant would target the amplification phase without interfering with initiation or termination, in order to allow some hemostasis in the event of tissue injury. Activated factor Xa (FXa) is central to the coagulation cascade and is the cornerstone of serine protease activity amplification. FX is a vitamin K-dependent serine protease synthesized in the liver that can be activated by either the intrinsic or the extrinsic clotting cascade. Binding of FXa to activated FV in the presence of calcium on a phospholipid bilayer results in formation of the prothrombinase complex. FXa catalyzes the conversion of prothrombin (Factor II) to thrombin (Factor IIa) and is the rate-limiting step in thrombin generation (Fig. 6.1) (28). One molecule of FXa can catalyze the formation of over a thousand molecules of thrombin (29). Thrombin potentiates clot formation by

Oral Direct Factor Xa Inhibitors

Intrinsic pathway

185

Extrinsic pathway

FXa

FXa

+ AT Prothrombin

Thrombin

FX

AT

Fibrin clot Fibrinogen

Fig. 6.1. Rivaroxaban selectively inhibits FXa. Schematic representation of the mechanism of inhibition of FXa by rivaroxaban

up-regulating its own production through feedback activation of factors V, VII, VIII, and XI, and inducing platelet activation (30, 31). Therefore, inhibition of FXa represents a potentially more efficacious anticoagulation strategy than targeting all of the vitamin K-dependent clotting factors. Inhibition of FXa can occur through direct binding to the FXa active site or indirectly through interaction with antithrombin. Direct FXa inhibitors have an advantage because they can bind both free FXa and FXa within the prothrombinase complex, and therefore penetrate the active thrombus to limit further thrombin generation.

2. Clinical Pharmacology of Rivaroxaban

Rivaroxaban is an oxazolidinone derivative that binds to the active site of FXa, leading to potent and selective inhibition of FXa (32). In animal models of both venous stasis and thrombosis, oral rivaroxaban inhibited FXa activity, leading to reduced thrombus formation and extension (33, 34). Rivaroxaban inhibits FXa activity in a dose-dependent manner, accompanied by prolongation of prothrombin time (PT) (Fig. 6.2). Phase-I data has shown that 15 mg of rivaroxaban decreases FXa activity by 35% and increases PT 1.4-fold over baseline values (35). The observed prolongation in PT correlated strongly with plasma rivaroxaban concentration (r = 0.935), with little inter-individual variability (36, 37). Thus, therapeutic monitoring might be possible through determination of PT when necessary. In phase-I studies in healthy male volunteers, a single 30-mg dose of rivaroxaban inhibited thrombin generation for greater than 24 h (38). Finally, rivaroxaban has no direct effect on platelet aggregation.

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40

PT Model

Prothrombin time (s)

30

20

10

0 0

100

200 300 400 Plasma concentration of BAY 59-7939 (µg/l)

500

600

Fig. 6.2. Prothrombin time correlates strongly with plasma concentration of rivaroxaban (r = 0.958). This figure reprinted with kind permission from Springer Science and Business Media (39).

2.1. Pharmacokinetics

The pharmacokinetic profile of rivaroxaban is consistent with rapid oral absorption and 80% bioavailability (36). The time to peak plasma concentration ranges from 2.5 to 4 h. After multiple doses, the drug half-life is 5–9 h in healthy volunteers, and 9–13 h in the elderly (mean age 65) (39). There are no major active circulating metabolites of rivaroxaban. A portion of the drug is excreted in the urine (1/3), and the remainder (2/3) is metabolized by the liver (36). Drug elimination demonstrates first-order kinetics and is impaired with advancing age, renal insufficiency, and in the presence of strong cytochrome P450 3A4 inhibitors (such as ketoconazole, macrolide antibiotics such as clarithromycin, and many protease inhibitors). In a phase-I study of patients with renal insufficiency, subjects with severe renal impairment (creatinine clearance <30 ml/min) experienced a 64% increase in serum drug concentration (p < 0.05 for AUC) and a 144% prolongation of PT (p < 0.001) compared to control subjects (40). Accordingly, patients with a creatinine clearance less than 30 ml/min, significant liver disease, and those taking strong cytochrome P450 3A4 inducers or inhibitors are being excluded from phase-III trials of rivaroxaban. While many anticoagulants are dose adjusted for extremes of body weight due to increased risks of bleeding, a phase-I randomized placebo, parallel group study of rivaroxaban in patients with extremes of body weight (≤50 kg or >120 kg) demonstrated no change in peak serum concentrations in those >120 kg, but did show mild elevation (24%) in those ≤50 kg. This minor elevation was associated with a small (15%) increase in PT, which was not considered clinically significant (41). Therefore, no dose adjustment is required for sex or body weight when dosing rivaroxaban.

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While the available phase-I data have been largely limited to healthy Caucasian males, studies examining rivaroxaban in raceand ethnicity-specific populations are ongoing. 2.2. Pharmacodynamic Interactions

Rivaroxaban absorption is improved when taken with food; however, the drug can be administered on an empty stomach. The pharmacokinetics of the drug are unaffected by ranitidine or alteration of gastric pH with antacids in healthy male volunteers (42). Many patients who will require oral anticoagulation for AF or VTE prophylaxis often have risk factors for or documented coronary artery disease and require aspirin therapy. Rivaroxaban is being studied in patients with acute coronary syndromes, a patient population treated with aspirin in addition to other antiplatelet agents. In a randomized two-way crossover study (with healthy male subjects), antiplatelet therapy with aspirin did not alter the pharmacokinetics or pharmacodynamics of rivaroxaban (as determined by bleeding and PTs). Furthermore, platelet aggregometry studies were unaffected by rivaroxaban (43). A similar phase-I, two-way crossover study demonstrated no clinically significant interaction between naproxen and rivaroxaban in healthy subjects (35). Since rivaroxaban might have a role in stroke prophylaxis in those with AF, 20 mg of rivaroxaban was co-administered in 20 healthy male volunteers who also received 0.375 mg of digoxin. Drug exposure was not significantly different between patients receiving rivaroxaban alone and those who received rivaroxaban and digoxin. Based upon these results, there appears to be no apparent interaction between rivaroxaban and digoxin, suggesting that they can be prescribed together (44). Bridging with low-molecular weight heparins (LMWHs) is common in patients receiving chronic oral anticoagulants. When given together with rivaroxaban, enoxaparin resulted in additive inhibition of FXa activity and prolongation of bleeding times; however, coadministration of LMWH and rivaroxaban has been demonstrated (45). Overall, compared to the VKAs and other cardiovascular medications such as amiodarone, rivaroxaban has relatively low potential for substantial pharmacodynamic interactions, allowing for a wide range of concomitant pharmacotherapy.

2.3. Toxicity and Adverse Effects

Therapeutic anticoagulation always carries an attendant risk of bleeding, either due to errors in dosing and administration, occult pathology such as gastric ulceration, unrecognized bleeding diatheses, or urgent and emergency medical procedures. Therefore, there is great interest in and need for neutralizing agents in the event of significant bleeding. To address this concern, investigators explored the use of recombinant activated factor VII (rFVIIa) as a partial reversal agent for rivaroxaban. In a rat model of mesenteric hemorrhage, rFVIIa was administered after

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high-dose rivaroxaban (2 mg/kg). In the presence of high-dose, supratherapeutic rivaroxaban, 400 mcg/kg of rFVIIa reduced bleeding times by nearly 50% (46). rFVIIa also partially reversed the prolongation of PT and partially restored total thrombin activity, without affecting rivaroxaban-dependent FXa inhibition. Therefore, rFVIIa might be of use as an intravenous antidote for major bleeding in patients taking rivaroxaban. While not yet R investigated, factor VIII inhibitor bypassing activity (FEIBA ), a vapor-heated lyophilized powder for reconstitution, also represents a potential alternative for serious and/or life-threatening bleeding with rivaroxaban. In 1,102 patients, rivaroxaban did not affect electrocardiographic parameters, including the QTc (47). This is important, as many patients with AF who might be candidates for rivaroxaban therapy will be taking antiarrhythmic drugs that are known to prolong the QT interval, including dofetilide and sotalol. Given the hepatotoxicity observed with ximelagatran (even though ximelagatran is a direct thrombin inhibitor, not a FXa inhibitor), particular attention has been focused on liver function surveillance in patients receiving rivaroxaban. In the recent Oral Direct Factor Xa Inhibitor BAY 59-7939 in Patients with Acute Symptomatic Deep-Vein Thrombosis (ODIXa-DVT) trial, there was no evidence of hepatotoxicity with long-term (3 months) administration of rivaroxaban for treatment of deep venous thrombosis (DVT) (48). In the first 3 weeks of this trial, patients randomized to rivaroxaban had a lower incidence of ALT >3×ULN (1.9–4.3% versus 21.6%) as compared to those receiving enoxaparin. After 3 weeks however, the incidence of ALT >3×ULN was similar in both groups [1.9% (95% CI 0.8–3.6) versus 0.9% (95% CI 0.0–4.8)]. Rivaroxaban was stopped early in three patients due to abnormal liver function tests (two of these patients died, one from fulminant hepatitis B and one from carcinoma with hepatic metastasis) (48). In a pooled analysis of 1,343 patients randomized in phase-II studies of rivaroxaban for the prevention of post-operative VTE, there was no difference in the incidence of ALT >3×ULN between rivaroxaban and enoxaparin (3.8–6.0% versus 7.7%) (47). While there is no evidence of increased hepatotoxicity with rivaroxaban in multiple phase-II studies or in early reports of phase-III studies of VTE prophylaxis, more long-term data are needed.

3. Clinical Indications As evidenced by the significant clinical and economic burden imposed by VTE, including DVT and pulmonary embolism (PE), in both medical and surgical patients, the rising incidence of AF in

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the rapidly expanding elderly population, and the global impact of ischemic heart disease, there are many potential patient populations and indications for novel, safe, and effective oral anticoagulants such as rivaroxaban. Accordingly, a broad network of clinical trials has been designed to evaluate the safety and efficacy of rivaroxaban in patients at risk for arterial and venous thrombosis. In the sections to follow, we will review completed, ongoing, and planned clinical trials of rivaroxaban, according to clinical indication.

4. Post-operative Thromboprophylaxis

Proof of principle for the use of rivaroxaban in the prevention of VTE was first demonstrated in a phase-IIa study of 642 patients undergoing total hip replacement (ODIXa-HIP). In this open-label, dose-escalation (12-fold dose range) study, patients were randomized in a 3:1 ratio to rivaroxaban (2.5, 5, 10, 20, and 30 mg twice daily, or 30 mg once daily) or enoxaparin (40 mg daily) (49). Patients were treated until mandatory bilateral venography was performed 5–9 days after surgery. The primary efficacy endpoint (DVT, PE, or all-cause mortality) was not different in those treated with rivaroxaban compared to enoxaparin (10.2–23.8% vs. 16.8%). There was a dose-dependent reduction in major VTE (defined as proximal DVT, PE, VTE-related death, Table 6.1) with rivaroxaban, which was accompanied by a dose-dependent (0–10.8%) increase in the incidence of major post-operative bleeding (P<0.001). While this study successfully demonstrated proof of concept for the therapeutic efficacy of rivaroxaban, it was limited by the open-label design and inadequate power to compare efficacy between enoxaparin and individual rivaroxaban doses. The ODIXa-KNEE trial was a randomized double-blind, double-dummy phase-IIb dose-ranging study in 621 patients undergoing total knee replacement (50). There was no difference between rivaroxaban (2.5, 5, 10, 20, 30 mg twice daily) and enoxaparin (30 mg twice daily) for the prevention of DVT, PE, and death. As previously observed, rivaroxaban was associated with a dose-dependent increase in major bleeding, but there was no difference when compared to enoxaparin. In the companion ODIXa-HIP2 trial, patients were randomized to the same escalating doses of rivaroxaban or enoxaparin 40 mg daily. ODIXaHIP2, like ODIXa-KNEE, was a double-blind, double-dummy trial with treatment until mandatory venography 5–9 days after surgery. As observed in the ODIXa-KNEE trial, there was no difference or dose effect on the composite endpoint (DVT, PE,

11.1

Major VTE (% proximal DVT, PE, and VTE-related death)

od = once a day, bid = twice a day

0

22.2

Primary endpoint (% DVT, PE, or all-cause mortality)

Major bleeding (%)

2.5 mg bid

Dose

2.5

7.9

23.8

5 mg bid

2.9

3.6

20.0

10 mg bid

Rivaroxaban (dose and regimen)

6.5

0

10.2

20 mg bid

10.8

4.3

17.4

30 mg bid

4.5

1.4

15.1

30 mg od

0

4.7

16.8

40 mg od

<0.001

0.01

0.05

P-value for Enoxaparin dose–response

Table 6.1 Safety and efficacy of rivaroxaban: phase-IIa study of VTE prophylaxis following total hip replacement

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or death) between rivaroxaban and enoxaparin. Again, there was a dose-dependent increase in major bleeding with rivaroxaban, but there was no difference compared to daily enoxaparin. In a pooled analysis of both studies (ODIXa-KNEE and ODIXaHIP2), which randomized a total of 1,343 patients (Table 6.2), there was a flat dose–response relationship for the prevention of total VTE with rivaroxaban (16.1–24.4% vs. 27.8% as compared to enoxaparin) (47). Not surprisingly, rivaroxaban was associated with a dose-dependent increase in incidence of major bleeding (defined as bleeding leading to death, bleeding into a critical organ such as the CNS, transfusion requirement >2 units, >2 g/dl fall in hemoglobin, or re-operation). The incidence of major bleeding ranged from 0.9 to 7% with rivaroxaban as compared to 1.7% with enoxaparin. This dose-dependent risk of bleeding remained significant, even after adjustment for age, gender, and study-specific bleeding rates. The majority of major bleeding episodes, as expected, were confined to the operative site. There was no significant difference in the risk of major bleeding between enoxaparin and rivaroxaban (total daily dose of 5–20 mg). Accordingly, this dose range was suggested as the optimal dosing for the prevention of VTE after orthopedic surgery. While ODIXa-KNEE and ODIXa-HIP2 compared twicedaily dosing of rivaroxaban, a randomized double-blind, doubledummy, active-comparator controlled trial of 873 patients examined once-daily rivaroxaban (over an eightfold dose range comprised of 5, 10, 20, 30, or 40 mg) compared to enoxaparin 40 mg daily for the prevention of VTE after total hip replacement (ODIXa-OD-HIP) (51). As in prior trials, patients were treated for 5–9 days after surgery and then underwent mandatory bilateral venography. In this once-daily dosing trial, there was a trend to significance in the dose-dependent reduction of VTE in patients treated with rivaroxaban. Perhaps of greater relevance was the statistically significant dose-dependent reduction in major VTE (proximal DVT, PE, or death). There was a dosedependent increase in the risk of major bleeding, however, the strength of the relationship was less than previously observed in the twice-daily dosing studies (2.3–5.1%, P = 0.039). Rivaroxaban was well tolerated in this trial, and no dose arm was stopped due to safety concerns. Based on the comparable efficacy between doses and the increase in major bleeding from 0.7 to 4.3% in the 10 mg as compared to 20 mg groups, the authors recommended 10 mg as the daily dose for future phase-III VTE prevention trials. All of these early trials led to the phase-III Regulation of Coagulation in Major Orthopaedic Surgery Deducing the Risk of

0.9

Major bleeding

1.3

2.4

2.1

3.1

∗ P-value for dosing trend; enox, enoxparin qd

3

10

30

7.0 <0.001

3.9

0.46

1.1

3.2

20

P-value∗ 21.6 22.9 16.1 24.4 19.3 0.39

5

1.7

4.5

2.3

8.5

0.7 4.3

2.7 0.9

40 mg 5 10 20 enox. 27.8 14.9 10.6 8.5

40

4.9 5.1

1.9 1.1

13.5 6.4

30

Once-daily dosing

Twice-daily dosing (bid)

2.5

ODIXa-OD-HIP (n=873)

ODIXa-HIP2 and KNEE pooled (n=1,343)

Major VTE (proximal DVT, PE, or VTE-related mortality)

Total VTE (DVT, PE, and all-cause mortality)

Dose (mg)

Trial

Table 6.2 Rivaroxaban dosing in thromboprophylaxis trials

0.039

0.007

P-value∗ 0.085

1.9

2.8

0.6

1

40 mg 10 enox. 25.2 9.6

0.5

2.6

NS

0.01

40 mg enox. P-value∗ 18.9 <0.001

Once-daily dosing

RECORD3 (n=2,531)

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DVT and PE (RECORD3) trial, which randomized over 2,500 patients to treatment with either rivaroxaban 10 mg daily or enoxaparin 40 mg daily for the prevention of VTE after total knee replacement. In this trial, enoxaparin was used in the manner recommended in the European package insert for the drug. Treatment with rivaroxaban led to a 49% relative risk reduction in the composite primary endpoint of DVT, PE, or death when compared to enoxaparin (9.6 versus 18.9%, P<0.001) (52). There was also a 62% relative risk reduction in major VTE (the secondary endpoint) with rivaroxaban (1 versus 2.6%, P = 0.01). This trial validated the selection of the 10 mg dose for prophylaxis of VTE after surgery, as the incidence of major bleeding was comparable to that with enoxaparin (0.6 versus 0.5%, P = NS). In summary, RECORD3 demonstrated superior efficacy of rivaroxaban as compared to enoxaparin for the prevention of VTE, with similar bleeding rates among patients undergoing total knee replacement.

5. Treatment of Venous Thrombosis

While the trials discussed above examined short-term anticoagulation with rivaroxaban, the ODIXa-DVT phase-II trial was the first to evaluate the use of rivaroxaban for treatment of known thrombus and subsequent long-term anticoagulation. In ODIXaDVT, patients were randomized in a parallel group design to anticoagulation with rivaroxaban (10, 20, 30 mg twice daily or 40 mg once daily) or enoxaparin followed by oral VKA antagonist therapy. The primary endpoint was improvement in thrombotic burden at day 21 (as determined by quantitative compression ultrasonography) without recurrent symptomatic VTE or VTE-related death. After 12 weeks of treatment, there was no difference between rivaroxaban and enoxaparin for the primary endpoint of thrombus reduction and recurrent VTE events (43.8–59.2% versus 45.9%), nor was there a dose–response relationship with rivaroxaban (P = 0.67). In addition, there was no dose–response between rivaroxaban and the primary safety endpoint (major bleeding). This study demonstrated proof of concept that rivaroxaban could be used for the treatment of existing clots. In the companion phase-II study, which evaluated oncedaily dosing (EINSTEIN-DVT) with rivaroxaban, 543 patients with symptomatic DVT without associated symptomatic PE were randomized to rivaroxaban (20, 30, or 40 mg daily) or heparin/LMWH followed by a VKA for 12 weeks (53). The primary outcome (deteriorating thrombus burden at 12 weeks

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as reflected by compression ultrasonography, perfusion lung scan, and recurrent symptomatic VTE) occurred in 5.4–6.6% of rivaroxaban patients as compared to 9.9% of heparin/VKA patients. There was no dose–response effect observed with rivaroxaban for the primary endpoint or for clinically relevant bleeding (primary safety endpoint) (2.9–7.5% versus 8.8% for heparin/VKA). The results of EINSTEIN-DVT, when viewed in the context of the thromboprophylaxis trials and ODIXa-DVT, suggest that dose-dependent bleeding risk is most evident in twice-daily dose regimens as compared to once-daily dosing (see Table 6.2). The results of ODIXa-DVT and EINSTEIN-DVT, which randomized 1,156 patients, demonstrated that rivaroxaban can effectively reduce thrombus burden in patients with DVT, with an acceptable safety profile. Nonetheless, phase-III studies using hard clinical endpoints are required before long-term anticoagulation with rivaroxaban can be advocated in clinical practice. At present, phase-III studies evaluating the use of rivaroxaban for the treatment of PE (EINSTEIN-PE) and long-term anticoagulation in patients with DVT or PE who have already received 6–12 months of oral rivaroxaban or VKA therapy (EINSTEINExtension) are currently recruiting patients.

6. Ongoing Phase-II–III Studies in Cardiovascular Disease

6.1. Rivaroxaban in ACS

Cardiovascular disease remains the leading cause of death in the United States. Approximately one million Americans suffer a myocardial infarction each year. By the year 2050, it is estimated that over 5.6 million Americans will have AF, the most common heart rhythm disorder encountered in clinical practice. There is great interest in the need for novel oral anticoagulants for the prevention of thromboembolic events in patients with AF as well as the prevention of recurrent myocardial infarction and death in patients with acute coronary syndromes (ACS). Rivaroxaban, with its associated safety and ease of dosing, is an attractive candidate for anticoagulation for prevention of stroke in AF and secondary prevention in patients with prior myocardial infarction. The Anti-Xa Therapy to Lower Cardiovascular Events in Addition to Aspirin with or without Thienopyridine Therapy in Subjects with Acute Coronary Syndrome (ATLAS ACS TIMI 46) trial is a phase-II placebo-controlled randomized study designed to evaluate the safety of rivaroxaban in patients with recent ACS (www.clinicaltrials.gov; IdentifierNCT00402597). The trial will enroll patients between the ages of 18 and 75 who have symptoms

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195

suggestive of ACS, a diagnosis of ST segment elevation or non-ST segment elevation myocardial infarction within the past 7 days, and at least one additional high-risk feature. Patients are being randomized to placebo, daily rivaroxaban, or twice-daily rivaroxaban. The trial will have two stages. The primary endpoint of the dose-escalation stage is significant TIMI bleeding. In the subsequent dose confirmation phase, the primary endpoint will be a composite of major adverse cardiac events (including death, recurrent myocardial infarction, stroke, or recurrent ischemia requiring revascularization). Patient randomization will be stratified according to the presence or absence of thienopyridine treatment in order to assess the risk benefit of anti-FXa activity with mono or dual antiplatelet therapy. 6.2. Rivaroxaban for Stroke Prophylaxis in Nonvalvular AF

The Rivaroxaban Once-Daily Oral Direct Factor Xa Inhibition Compared with Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation (ROCKET-AF) trial is a prospective, randomized, double-blind, double-dummy, parallel group, multicenter, event-driven, non-inferiority study comparing the efficacy and safety of once-daily oral rivaroxaban with adjusted dose oral warfarin for the prevention of stroke and non-CNS embolism in patients with non-valvular AF. In ROCKET-AF, patients are being randomized to rivaroxaban 20 mg once-daily (15 mg if creatinine clearance is 30–49 ml/min) versus dose-adjusted warfarin. Blinded treatment will be maintained through the use of a double-dummy system including sham INRs in patients receiving rivaroxaban. The primary efficacy endpoint for the trial is a composite of stroke or nonCNS embolism. The objective of the primary efficacy analysis is to establish that rivaroxaban is not inferior to warfarin. The challenges of non-inferiority trials against warfarin are well documented (54, 55). Given the low frequency of stroke in patients receiving dose-adjusted warfarin, a non-inferiority trial will require a large sample size. ROCKET-AF will randomize at least 14,000 patients in order to test the hypothesis. The primary safety endpoint of ROCKET-AF is the composite of major and non-major clinically relevant bleeding episodes. Secondary endpoints will include the individual components of the primary endpoints, in addition to myocardial infarction, disabling stroke, and all-cause mortality.

7. Expert Opinion There is a clinical need for new oral anticoagulants. FXa represents an attractive pharmacologic target for new agents. While efficacy is paramount, so too is safety, given the morbidity and mortality

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associated with bleeding, especially in the predominantly elderly population in whom oral anticoagulants are prescribed. The drug development plan for rivaroxaban is aggressive, with simultaneous investigative programs spanning multiple indications rather than a sequential approach. At present, over 24,000 patients (12,000 are predominantly post-operative orthopedic patients) have been evaluated in completed phase-II and III trials of rivaroxaban for thromboprophylaxis and treatment of DVT. By the time all the currently enrolling trials have concluded, more than 50,000 patients will have been evaluated in all randomized controlled trials of rivaroxaban. The advantages of rivaroxaban include the potential for oncedaily dosing for all indications, no required dose-adjustment for body weight, no known interactions with common cardiovascular medications, a relatively safe pharmacodynamic profile with respect to bleeding risk and hepatotoxicity, no clinically significant interaction with aspirin, and the ability to bridge with LMWH when necessary. On the other hand, rivaroxaban is partially renally cleared and will require dose adjustment in those with grade-III chronic kidney disease, and is not being studied in patients with a creatinine clearance less than 30 ml/min. Since rivaroxaban metabolism is affected by potent cytochrome P450 3A4 inhibitors such as ketoconazole, clarithromycin, and protease inhibitors, its use will be restricted in some special populations. Nonetheless, after extensive phase II, and now emerging phase III trial data, it appears that rivaroxaban is effective in preventing and treating VTE with a bleeding risk comparable to other anticoagulants. The results of randomized trials evaluating rivaroxaban for the prevention of stroke and non-CNS embolism in AF and secondary prevention of ACS are currently ongoing. While there are several other oral anticoagulants in development, none have been evaluated as extensively and in as many patients as rivaroxaban (Table 6.3). Rivaroxaban also has the advantage that it can be dosed once a day, which has been shown to improve patient compliance and outcomes (56, 57). Despite once-daily dosing, rivaroxaban has a half-life that is considerably shorter than other oral FXa inhibitors, which is an advantage in the event of bleeding or an urgent need to discontinue anticoagulation. Unlike the direct thrombin inhibitor dabigatran, bioavailability of rivaroxaban is very good, and it has low potential for drug–drug interactions, including medications which alter gastric pH that are taken chronically by 3% of the US population (58). Perhaps more importantly, rivaroxaban has been shown to have no effect on platelet aggregation and its pharmacokinetic profile is unaffected by aspirin and other notable cardiovascular medications, such as digoxin. Finally, after clinical investigation in

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Table 6.3 Some alternative oral anticoagulants in phase-III trials Rivaroxaban (BAY 59-7939)

Apixaban

Dabigatran (BIBR 1048)

Target

Factor Xa

Factor Xa

Factor IIa (thrombin)

Dosing

Once or twice daily

Twice daily

Once or twice daily

Bioavailability (%)

80

24–88

4

Half-life (h)

5–9 and 11–13 in the elderly

Metabolism (%) Renal Hepatic Adjustments and interactions

9–14

8–17

33 (unchanged) 67

25 75

80 20

• Avoid with CrCl <30 ml/min • Dose reduction for CrCl 30–49 ml/min • Avoid strong CYP450 3A4 inhibitors

• Avoid strong CYP450 3A4 inhibitors

• Avoid proton pump inhibitors (↓ absorption by 20–25%)

thousands of patients, rivaroxaban appears to have no significant hepatotoxicity and a bleeding risk comparable to other conventional anticoagulants.

8. Conclusion Rivaroxaban is a novel, oral direct FXa inhibitor that does not require any cofactors. This therapeutic has potential use for several clinical indications and disease states, including VTE prophylaxis, long-term treatment of DVT, PE, and the prevention of stroke and non-CNS embolism in patients with AF, and potentially ACS. In order to understand the efficacy and safety of rivaroxaban across such a wide range of patient populations, several large simultaneous studies evaluating rivaroxaban in both venous and arterial thromboembolism are under way. Rivaroxaban might have its greatest impact in providing a much-needed and attractive alternative to warfarin. While formal cost-effective analyses are not yet available, avoidance of the intensive, costly, and frequent monitoring required with VKAs, as well as the potential to reduce adverse vascular events precipitated by the narrow therapeutic window of VKAs, should result in a significant improvement in quality of life and cost savings. While further data (particularly

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large phase-III trials) and caution are required, there is reason for optimism. Rivaroxaban may very well be the long-awaited alternative to warfarin. References 1. Rosamond, W., Flegal, K., Friday, G., Furie, K., Go, A., Greenlund, K., Haase, N., Ho, M., Howard, V., Kissela, B., Kittner, S., Lloyd-Jones, D., McDermott, M., Meigs, J., Moy, C., Nichol, G., O’Donnell, C.J., Roger, V., Rumsfeld, J., Sorlie, P., Steinberger, J., Thom, T., Wasserthiel-Smoller, S., Hong, Y., and for the American Heart Association Statistics Committee and Stroke Statistics, S. (2007) Heart disease and stroke statistics– 2007 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee Circulation 115, e69–171. 2. Caprini, J.A., Botteman, M.F., Stephens, J.M., Nadipelli, V., Ewing, M.M., Brandt, S., Pashos, C.L., and Cohen, A.T. (2003) Economic burden of long-term complications of deep vein thrombosis after total hip replacement surgery in the United States Value Health 6, 59–74. 3. MacDougall, D.A., Feliu, A.L., Boccuzzi, S.J., and Lin, J. (2006) Economic burden of deep-vein thrombosis, pulmonary embolism, and post-thrombotic syndrome Am J Health Syst Pharm 63, S5–15. 4. Go, A.S., Hylek, E.M., Phillips, K.A., Chang, Y., Henault, L.E., Selby, J.V., and Singer, D.E. (2001) Prevalence of diagnosed atrial fibrillation in adults: national implications for rhythm management and stroke prevention: the anticoagulation and risk factors in atrial fibrillation (ATRIA) study JAMA 285, 2370–5. 5. Wolf, P.A., Abbott, R.D., and Kannel, W.B. (1991) Atrial fibrillation as an independent risk factor for stroke: the Framingham Study Stroke 22, 983–8. 6. Dulli, D.A., Stanko, H., and Levine, R.L. (2003) Atrial fibrillation is associated with severe acute ischemic stroke Neuroepidemiology 22, 118–23. 7. Link, K. (1959) The discovery of dicumarol and its sequels Circulation 19, 97–107. 8. Hart, R.G., Pearce, L.A., Aguilar, M.I. (2007) Meta-analysis: antithrombotic therapy to prevent stroke in patients who have nonvalvular atrial fibrillation Ann Intern Med 146, 857–67. 9. The ACTIVE Writing Group on behalf of the ACTIVE investigators (2006) Clopidogrel plus aspirin versus oral anticoagulation

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Oral Direct Factor Xa Inhibitors 18. Stroke Prevention in Atrial Fibrillation Investigators (1994) Warfarin versus aspirin for prevention of thromboembolism in atrial fibrillation: stroke prevention in Atrial Fibrillation II Study Lancet 343, 687–91. 19. Fang, M.C., Go, A.S., Hylek, E.M., Chang, Y., Henault, L.E., Jensvold, N.G., and Singer, D.E. (2006) Age and the risk of warfarin-associated hemorrhage: the anticoagulation and risk factors in atrial fibrillation study J Am Geriatrics Soc 54, 1231–6. 20. McCormick, D., Gurwitz, J.H., Goldberg, R.J., Becker, R., Tate, J.P., Elwell, A., and Radford, M.J. (2001) Prevalence and quality of warfarin use for patients with atrial fibrillation in the long-term care setting Arch Intern Med 161, 2458–63. 21. Fang, M.C., Stafford, R.S., Ruskin, J.N., and Singer, D.E. (2004) National trends in antiarrhythmic and antithrombotic medication use in atrial fibrillation Arch Intern Med 164, 55–60. 22. Olsson, S.B. (2003) Stroke prevention with the oral direct thrombin inhibitor ximelagatran compared with warfarin in patients with non-valvular atrial fibrillation (SPORTIF III): randomised controlled trial Lancet 362, 1691–8. 23. Albers, G.W., Diener, H.C., Frison, L., Grind, M., Nevinson, M., Partridge, S., Halperin, J.L., Horrow, J., Olsson, S.B., Petersen, P., and Vahanian, A. (2005) Ximelagatran vs warfarin for stroke prevention in patients with nonvalvular atrial fibrillation: a randomized trial Jama 293, 690–8. 24. Lee, W.M., Larrey, D., Olsson, R., Lewis, J.H., Keisu, M., Auclert, L., and Sheth, S. (2005) Hepatic findings in long-term clinical trials of ximelagatran Drug Safety 28, 351–70. 25. Boos, C.J., and Lip, G.Y.H. (2006) Ximelagatran: an eulogy Thromb Res 118, 301–4. 26. van Walraven, C., Hart, R.G., Singer, D.E., Laupacis, A., Connolly, S., Petersen, P., Koudstaal, P.J., Chang, Y., and Hellemons, B. (2002) Oral anticoagulants vs aspirin in nonvalvular atrial fibrillation: an individual patient meta-analysis JAMA 288, 2441–8. 27. Rosenberg, R.D., and Aird, W.C. (1999) Vascular-bed – specific hemostasis and hypercoagulable states N Engl J Med 340, 1555–64. 28. Bauer, K.A. (2006) New anticoagulants: anti IIa vs anti Xa – is one better? J Thromb Thrombolysis 21, 67–72. 29. Mann, K.G., Brummel, K., and Butenas, S. (2003) What is all that thrombin for? J Thromb Haemost 1, 1504–14.

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30. Suzuki, K., Dahlback, B., and Stenflo, J. (1982) Thrombin-catalyzed activation of human coagulation factor V J Biol Chem 257, 6556–64. 31. Walsh, P.M.D. (2004) Platelet coagulationprotein interactions Sem Thromb Hemost 461–71. 32. Roehrig, S., Straub, A., Pohlmann, J., Lampe, T., Pernerstorfer, J., Schlemmer, K.H., Reinemer, P., and Perzborn, E. (2005) Discovery of the novel antithrombotic agent 5-chloro-N-({(5S)-2-oxo-3- [4-(3oxomorpholin-4-yl)phenyl]-1,3-oxazolidin5-yl}methyl)thiophene- 2-carboxamide (BAY 59-7939): an Oral, Direct Factor Xa Inhibitor J Med Chem 48, 5900–8. 33. Biemond, B.J., Perzborn, E., Friederich, P.W., Levi, M., Buetehorn, U., and Buller, H.R. (2007) Prevention and treatment of experimental thrombosis in rabbits with rivaroxaban (BAY 597939) – an Oral, Direct Factor Xa Inhibitor Thromb Haemost 97, 471–7. 34. Perzborn, E., Strassburger, J., Wilmen, A., Pohlmann, J., Roehrig, S., Schlemmer, K.H., and Straub, A. (2005) In vitro and in vivo studies of the novel antithrombotic agent BAY 59-7939 – an Oral, Direct Factor Xa Inhibitor J Thromb Haemost 3, 514–21. 35. Kubitza, D., Becka, M., Mueck, W., and Zuehlsdorf, M. (2007) Rivaroxaban (BAY 59-7939) – an Oral, Direct Factor Xa Inhibitor – has no clinically relevant interaction with naproxen Br J Clin Pharmacol 63, 469–76. 36. Kubitza, D., Becka, M., Voith, B., Zuehlsdorf, M., and Wensing, G. (2005) Safety, pharmacodynamics, and pharmacokinetics of single doses of BAY 59-7939, an Oral, Direct Factor Xa Inhibitor Clin Pharmacol Ther 78, 412–21. 37. Mueck, W., Becka, M., Kubitza, D., Voith, B., and Zuehlsdorf, M. (2007) Population model of the pharmacokinetics and pharmacodynamics of rivaroxaban – an Oral, Direct Factor Xa Inhibitor – in healthy subjects Int J Clin Pharmacol Ther 45, 335–44. 38. Hader, S., Graff, J., Hentig, N., Misselwitz, F., Kubitza, D., Zuelsdorf, M., Wensing, G., Mueck, W., Becka, M., and Breddin, H.-K. (2003) Effects of BAY 597939, an Oral, Direct Factor Xa Inhibitor, on thrombin generation in healthy volunteers Blood 102. 39. Kubitza, D., Becka, M., Wensing, G., Voith, B., and Zuehlsdorf, M. (2005) Safety, pharmacodynamics, and pharmacokinetics of BAY 59-7939—an Oral, Direct Factor Xa Inhibitor—after multiple dosing in healthy

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Oral Direct Factor Xa Inhibitors 57. Richter, A., Anton, S.E., Koch, P., and Dennett, S.L. (2003) The impact of reducing dose frequency on health outcomes Clin Ther 25, 2307–35; discussion 6.

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Chapter 7 Antiplatelet Therapies: Drug Interactions in the Management of Vascular Disorders Shaker A. Mousa Abstract Antiplatelet drugs represent a key class of drugs that are of proven value in arterial thromboembolic disorders. There is a need for effective, safe antiplatelet agents or their combinations to provide predictable therapeutic benefit, dosage flexibility, and unique pharmacologic profiles, such as rapid onset in acute thrombotic states, as well as sustained antiplatelet effects in chronic platelet-activating states (e.g., post-stent placement). Aspirin, clopidogrel, or their combination have shown improved clinical outcomes in certain unique settings, and the search for additional antiplatelet agents is ongoing. Current studies suggest that combination antiplatelet therapy with existing agents is best considered a use-adapted strategy, with the greatest clinical benefit of combination therapy realized in acute, platelet-activating, and prothrombotic states. Key words: Acute coronary syndrome, clopidogrel, aspirin, myocardial infarction, antiplatelets, combination therapy, ADP receptor antagonist, percutaneous coronary intervention.

1. Introduction Platelets play a key role in arterial thrombosis, where platelet activation and aggregation are the proximate events associated with acute coronary syndrome (ACS), stroke, and peripheral artery disease (PAD) (1–6). Platelet adhesion to injured vascular endothelium leads to platelet activation, which is further amplified by various platelet agonists, including arachidonic acid, ADP, thrombin, serotonin, and collagen (5–7). Aspirin was the first antiplatelet drug to provide insight into the role of platelets in health and disease (6, 8). Since its development in 1899 and the subsequent elucidation of its mechanism of action, S.A. Mousa (ed.), Anticoagulants, Antiplatelets, and Thrombolytics, Methods in Molecular Biology 663, DOI 10.1007/978-1-60761-803-4_7, © Springer Science+Business Media, LLC 2003, 2010

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thromboxane A2 blockade has become one of the most wellknown antiplatelet paradigms (8). Aspirin inhibits the formation of thromboxane by irreversibly inhibiting COX-1 via acetylation of the serine-529 peptide, the requisite site for the formation of thromboxane from arachidonic acid. It was not until the late 1970s that aspirin was widely recognized for its therapeutic benefit in cardiovascular disease (2, 9). Aspirin is known to reduce the risk of myocardial infarction (MI), lower the risk of developing ischemic stroke, and decrease mortality in patients with vascular disease (10). Aspirin is presently recommended for the management of acute MI and unstable angina and is an effective therapeutic agent for secondary prevention of ischemic events (11). The development of the first ADP receptor antagonist, ticlopidine, followed the recognized clinical benefit of aspirin (12). Ticlopidine also demonstrated antithrombotic benefits but was associated with a high incidence of neutropenia, aplastic anemia, and thrombotic thrombocytopenic purpura (13). The discovery of a second thienopyridine ADP receptor antagonist, clopidogrel, confirmed the promise of antiplatelet therapy via alteration of the platelet functional pathway. Clopidogrel was comparable to ticlopidine in terms of efficacy, but had a better safety profile (14, 15) and was associated with a lower incidence of neutropenia and thrombotic thrombocytopenic purpura (16). With the development of various antiplatelet therapies, consideration was given to the therapeutic strategy of combining different antiplatelet agents (17, 18). One of the earliest examples of the R clinical success of such dual antiplatelet therapy was Aggrenox , a combination of a low-dose aspirin with dipyridamole for stroke prevention (19). While aspirin continues to be used routinely for the management of acute MI, unstable angina, and secondary prevention of ischemic events, dual antiplatelet therapy has been tested in several different clinical populations to achieve optimal efficacy with minimal adverse affects. For example, aspirin has been combined with clopidogrel in different doses and at different durations to assess its effectiveness as compared to aspirin alone (20) (Table 7.1). Improved efficacy has not always been the case, however, and in some cases, dual antiplatelet therapy carries a significant hemorrhagic risk (21). When contemplating a combination therapy, there are several factors that must be carefully weighed against the benefits. Each agent should individually demonstrate significant clinical benefits, and the combination of agents should have a greater benefit/risk ratio than either one alone. Ideally, each agent should act at a different mechanistic level and with different capacities. When combining the agents at different doses, one should be able to demonstrate improved efficacy without a compromise in safety.

Study population

3,491 patients, acute setting: patients had experienced ischemia within 12 h prior to treatment

45,852 patients, acute setting: patients suspected of acute MI

15,603 patients, non-acute setting: all patients were at high risk for atherothrombic events. Patients were further defined as either symptomatic (those with documented cardiovascular disease) or asymptomatic (those who only displayed risk factors)

Trial

CLARITY-TIMI

COMMIT

CHARISMA

Multi-center, randomized, double-blind, placebo-controlled. All patients received aspirin, and were then randomized to receive either clopidogrel or placebo. Duration: 28 months

Multi-center, randomized, double-blind, placebo-controlled. All patients received aspirin and were then randomized to either clopidogrel or placebo. Duration: discharge or 4 weeks

Multi-center, randomized, double-blind, placebo-controlled. All patients received aspirin and were then randomized to receive either clopidogrel or placebo. Duration: 30 days

Design

Patients in the symptomatic subgroup: risk rate reduction of 1% in primary end points with combination therapy as compared to aspirin alone. Patients in asymptomatic subgroup: 1% increase in the rate of primary end points when given combination therapy as compared to aspirin alone. No increase in major bleeding was seen with the addition of clopidogrel

Significant reduction (9%) of death and reinfarction were seen with combination therapy as compared to aspirin alone. Combination therapy was not beneficial in reducing stroke

Significant reduction (36%) of composite death, recurrent MI, and need for revascularization was seen with combination therapy as compared to aspirin alone. No significant increase in major bleeding was observed in the combination group

Outcome

Table 7.1 Summary of combination therapies with clopidogrel plus aspirin versus aspirin alone in different patient populations

Antiplatelet Therapies: Drug Interactions in the Management of Vascular Disorders 205

Study population

12,562 patients, acute setting: patients had received CABG and PCI interventions and had symptoms indicative of ACS with evidence of ischemia

7,599 patients, non-acute setting: patients had experienced ischemic stroke or TIA within the past 3 months and displayed additional risk factors

107 patients, acute setting: patients were recently symptomatic with >50% carotid stenosis, and ipsilateral carotid territory TIA or stroke within 3 months prior to treatment

Trial

CURE and PCICURE

MATCH

CARESS

Table 7.1 Continued

Multi-center, randomized, double-blind, placebo-controlled. All patients received aspirin and were then randomized to receive either clopidogrel or placebo. Microemboli were screened at 7 days with transcranial Doppler. Duration: 7 days

Multi-center, randomized, double-blind, placebo-controlled. Patients receiving clopidogrel were randomized to receive either aspirin or placebo Duration: 18 months

Multi-center, randomized, double-blind, placebo-controlled. All patients received aspirin, and were then randomized to receive either clopidogrel or placebo Duration: 1 year

Design

Significant reduction (39%) in the primary end point was seen with combination therapy as compared to aspirin alone

No significant difference was observed in the reduction of MI, vascular death, or acute ischemic events with combination therapy as compared to aspirin alone. Combination therapy group experienced a significantly higher rate of major bleeding

CURE: significant reduction (2%) in the primary end point composite of cardiovascular death, MI, or stroke was seen with combination therapy as compared to aspirin alone PCI-CURE sub-analysis: patients who received PCI intervention demonstrated a larger degree of relative risk reduction (4%) with no significant excess in major bleeding

Outcome

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2. Combination Antiplatelets: Issues and Perspective 2.1. Heparin and Platelet GPIIa/IIIa Antagonists

The most recent forms of antiplatelet therapy focus on fibrinogen displacement in existing thrombi to prevent further platelet crosslinking and thrombosis via the platelet GPIIb/IIIa complex (22–25). Abciximab, tirofiban, and eptifibatide are examples of GPIIb/IIIa receptor inhibitors that have shown clinical benefits in various trials (26–31) and have been incorporated into current American College of Cardiology/American Heart Association (ACC/AHA) treatment guidelines for unstable angina/nonST elevation MI (32). There is a wealth of clinical experience with the use of heparin and intravenous platelet GPIIb/IIIa antagonists in ACS and combinations of intravenous GPIIb/IIIa antagonists with thrombolytics. The potential clinical benefit of the platelet GPIIb/IIIa antagonist abciximab in ACS was demonstrated in the pivotal EPIC and EPILOG trials. In the EPIC trial, there was significant excess bleeding that occurred when unfractionated heparin (UFH) was used in its full dose with abciximab, leading to the EPILOG trial, where a reduced dose of heparin was used. The lower heparin dose led to an improved safety profile without compromising the efficacy observed in the EPIC trial. Similarly, the PRISM and the PRISM-PLUS trials evaluated whether administration of aspirin plus tirofiban (PRISM) or aspirin, heparin, and tirofiban (PRISM-PLUS) would improve clinical outcomes in the management of unstable angina. Tirofiban plus heparin was significantly more effective than tirofiban without heparin in reducing the incidence of death, MI, or refractory ischemia. Surprisingly, there was increased mortality with intravenous tirofiban without heparin, similar to that observed in the various oral GPIIb/IIIa antagonist trials, suggesting the critical need for heparin or anticoagulants with intravenous and perhaps oral GPIIb/IIIa antagonists. The interactions between platelet GPIIb/IIIa receptor antagonists and heparin or lowmolecular weight heparin (LMWH) could have tremendous clinical implications. LMWH may enhance the antiplatelet activity of GPIIb/IIIa antagonists by inhibiting thrombin, FXa, and other coagulation factors, along with inducing the vascular release of tissue factor pathway inhibitor (TFPI). At the same time, GPIIb/IIIa antagonists may potentiate the anticoagulant action of LMWH by blocking fibrinogen binding and aggregation, regardless of the activating stimulus and the down-regulation of pro-coagulant activity on the platelet surface. The combination of reduced dose LMWH or anticoagulant with a reduced

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dose GPIIb/IIIa antagonist and the combination of GPIIb/IIIa antagonist with thrombolytic may result in a higher therapeutic index in various thrombotic disorders and beyond. 2.2. Aspirin and Clopidogrel

This segment will focus on the rationale and current clinical status of dual antiplatelet therapy consisting of aspirin and clopidogrel (versus monotherapy with aspirin alone) in arterial vascular disease. The CLARITY-TIMI and COMMIT trials provided the basis for the use of aspirin plus clopidogrel for patients suffering from acute MI. In contrast, however, the CHARISMA trial demonstrated that the combination was not justified in stable high-risk patients with documented coronary disease, cerebrovascular disease, or symptomatic PAD. In two additional cardiovascular studies, the CURE and the PCI-CURE trials, the benefit of the drug combination was demonstrated in patients who underwent percutaneous coronary intervention (PCI) and coronary artery bypass graft (CABG). Finally, we will discuss two focused cerebrovascular studies, the CARESS and MATCH trials. The CARESS trial demonstrated the clinical benefit of the combination in an acute clinical setting. However, the combination proved to be ineffective in the longer term MATCH trial. It appears from these large clinical trials that the risk benefit of dual antiplatelet therapy with aspirin and clopidogrel is justified in high-risk symptomatic patients but not in asymptomatic patients. The exact dose regimen and duration of combination therapy await definition and require careful assessment to optimize the anticoagulant benefit while minimizing the hemorrhagic risk.

2.2.1. Limitations of Combined Therapy

Aspirin established the clinical benefit of antiplatelet agents, and clopidogrel exhibited augmented efficacy when combined with aspirin in certain, but not all clinical settings. However, these agents are not without shortcomings, and their limitations are a necessary backdrop to the studies discussed. Aspirin resistance, a phenomenon described clinically as a lack of desired response while the patient is receiving aspirin, is estimated to affect ≥20% of the population. Either on the basis of genetic polymorphism, noncompliance, or concomitant administration of a competing medication (e.g., an NSAID), aspirin resistance remains an uncontrolled variable without a laboratory definition (33, 34). Clopidogrel, a prodrug, suffers from delayed onset, a limitation that can be partially overcome by dose loading. The use of this strategy in some of the studies discussed below is noted. Other thienopyridines, such as prasugrel, which do not have this limitation, are currently in phase-III clinical trials (35). There is also evidence that the dose benefit of clopidogrel co-therapy may be disease related, as in the case of type II diabetes, in which 60% of the patients demonstrated platelet reactivity after receiving

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twice the standard maintenance dose of typical clopidogrel therapy (36). As with aspirin, clopidogrel noncompliance is also a serious clinical confounding element, and in the presence of drugeluting stents, is strongly associated with subsequent mortality. Regardless of the status of development of a given antiplatelet agent or combination therapy, full clinical benefit can only be realized by parallel strategies in patient education to ensure medication compliance (37). In the case of unstable angina and non-STsegment elevation MI, clinical guides detailing the use of aspirin and clopidogrel have been established. As mentioned earlier, a great concern of combination therapy is the increased risk for bleeding. In the MATCH (Management of Atherothrombosis with Clopidogrel in High-Risk Patients with Recent Transient Ischemic Attack or Stroke) trial (see Section 2.2.6), increased risk of bleeding, possibly associated with the duration of therapy, proved to be a significant issue. Finally, discussion of current dual antiplatelet therapy would be incomplete without the mention of combined therapy in the prevention of stent thrombosis, in which dual therapy is considered the standard of care. Combined antiplatelet therapy reduces stent thrombosis, and current ACC/AHA guidelines on PCI recommend clopidogrel plus aspirin (38). This is an area of great controversy regarding dose and duration of dual therapy, as guidelines were based on studies done with bare metal stents. In the current therapeutic era, most stents used are of the drug-eluting type in which delayed neointimal revascularization presents a risk for late (> 6 months) stent thrombosis. 2.2.2. The CLARITY-TIMI Study

The CLARITY-TIMI (Clopidogrel as Adjunctive Reperfusion Therapy-Thrombolysis in Myocardial Infarction 28) trial, a large trial that involved 28 centers, compared the use of clopidogrel with aspirin to aspirin alone (39). In this study, 3,491 patients who experienced ischemia lasting >20 min within the prior 12 h and associated ST-segment elevation or new-onset left-bundle branch block were scheduled to receive a fibrinolytic agent, aspirin, and heparin. The subjects were randomized to receive clopidogrel or placebo and were followed for 30 days. The composite end point was occlusion of the infarct-related artery or death from any cause prior to angiography. The primary safety end point was major hemorrhage defined by TIMI criteria after angiography. Patients in the clopidogrel-treatment group had a composite reduction in recurrence of thrombosis in the infarct-related artery, MI, or death with a risk reduction of 36% (P < 0.001) at 2–8 days and a 20% reduction at 30 days. There was no significant increase in major bleeding between the two groups or any of the subgroups. The CLARITY-TIMI study showed the combination of clopidogrel and aspirin to be superior to aspirin

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alone in decreasing the rate of death and recurrent thrombosis in infarct-related arteries. 2.2.3. The COMMIT Study

In the COMMIT (Clopidogrel and Metoprolol in Myocardial Infarction Trial) trial (40), 45,852 patients suspected of acute MI received aspirin (162 mg) and were randomized to aspirin plus placebo or aspirin plus clopidogrel. Patients also received metoprolol or placebo. Patients did not receive a loading dose of clopidogrel and were monitored for 28 days or until discharge, whichever came first. Primary end points included a composite of death, reinfarction, stroke, or death from any cause. Primary end points were reached in 9.2% of the clopidogrel group versus 10.1% in the placebo group, and there was a 0.9% risk reduction of death, reinfarction, and stroke (P < 0.002) in favor of the clopidogrel group. There was also a significant (7%) reduction in death of any cause. This was accomplished with no significant excess bleeding in the clopidogrel-treated patients or in patients aged >70 years, or those given fibrinolytic therapy. Based on this study, it was concluded that routine use of clopidogrel plus aspirin in patients with acute MI safely reduces mortality and major vascular events in a large range of patients, including those aged >70 years.

2.2.4. The CHARISMA Study

The CHARISMA (Clopidogrel for High Artherothrombotic Risk and Ischemic Stabilization, Management, and Avoidance) trial was a 28-month trial evaluating antiplatelet combination therapy versus aspirin monotherapy for both primary and secondary prevention of atherothrombotic events in 15,603 stable patients (41). All patients were categorized as high risk for atherothrombotic events and were randomized to clopidogrel 75 mg/day plus low-dose aspirin (75–162 mg/day) or low-dose aspirin plus placebo. The efficacy end point was a composite of MI, stroke, or death from cardiovascular disease. Patients were categorized into two subgroups: a symptomatic subgroup (12,153 patients), composed of those with documented cardiovascular disease (remote MI, stroke or symptomatic PAD) and an asymptomatic group (3,284 patients), who were enrolled with multiple atherothrombotic risk factors but without established atherosclerosis. The primary safety end point was any event of severe bleeding based on GUSTO (Global Utilization of Streptokinase and Tissue Plasminogen Activator for Occluded Coronary Arteries) criteria. Overall, treatment was discontinued in 20.4% of patients in the clopidogrel group and in 18.2% of the placebo group (P< 0.001) due to adverse events. The primary end point occurred in 6.8% of the clopidogrel group and in 7.3% of the placebo group, which was not significant (P< 0.22). Severe bleeding occurred in 1.7% of the clopidogrel group and in 1.3% of the

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placebo group (P< 0.09). The relative risk reduction in the symptomatic group received clopidogrel (6.9%) versus that placebo (7.9%) was 0.88 (P = 0.046). The results of the CHARISMA study indicated that only in the selected symptomatic group was there a suggestion of risk reduction that outweighed the risk of bleeding, and in the asymptomatic group with multiple risk factors, there was a suggestion of harm. Clopidogrel plus aspirin was not more effective than aspirin alone in reducing the rate of death from MI, stroke, or death from cardiovascular disease in stable patients in this long-term study. 2.2.5. The CURE and PCI-CURE Studies

The CURE (Clopidogrel in Unstable Angina to Prevent Recurrent Events) trial evaluated the benefits and risks of clopidogrel plus aspirin versus aspirin alone in patients with non-ST elevation ACS (42). A total of 12,562 patients were included in the trial; 2,072 underwent CABG intervention, 2,658 underwent a PCI, and 2,658 were managed medically. Patients were included in the study if they had symptoms indicative of ACS within the preceding 24 h without ST-segment elevations, supporting evidence of ischemia from their most recent electrocardiogram, and elevated concentrations of cardiac enzymes (including troponin) that were at least twice the reference range. All patients received aspirin 75–325 mg/day and were then randomized to receive clopidogrel 75 mg/day with a loading of 300 mg or placebo for 3–12 months. The primary outcome was a composite of cardiovascular death, MI, or stroke among patients who underwent CABG, PCI, or medical therapy. Overall, 10.6% of patients in the clopidogrel group and 12.5% of patients in the placebo group experienced one of the primary outcomes. This occurred with a relative risk of 0.84 (P = 0.001). For patients who underwent CABG treatment, primary outcomes occurred in 16.2% of the placebo group versus 14.5% in the clopidogrel group. Benefits were seen mainly in those patients who had received combination therapy before the procedure. For patients undergoing CABG, there was no significant trend of lifethreatening bleeding with clopidogrel, the use of which was confined to within 5 days of CABG surgery. According to the CURE trial, the benefits of starting clopidogrel with aspirin in non-ST MI outweighed the hemorrhagic risk, even in patients treated by CABG. Clopidogrel-treated patients also experienced reduced in-hospital refractory ischemia, recurrent angina, and heart failure. The CURE trial concluded that clopidogrel is beneficial in ACS patients whether or not they undergo revascularization. With regard to maximizing the clinical benefit and minimizing the hemorrhagic risk associated with clopidogrel and CABG, the results of the CURE trial suggested initiating dual therapy upon presentation and stopping clopidogrel 5 days before the CABG procedure.

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The PCI-CURE (Percutaneous Coronary InterventionClopidogrel in Unstable angina to Prevent Recurrent Events) trial (42), published in 2001, was a sub-analysis of the CURE trial in which 2,658 patients who were included in the CURE study and had undergone PCI were studied. Primary outcomes were cardiovascular death, MI, or urgent revascularization within 30 days of the intervention. PCI patients were given clopidogrel or placebo in combination with aspirin for 4 weeks prior to surgery. They resumed drug treatment post-surgically and were assessed for long-term effects up to 1 year later. There was a significant difference in cardiovascular death and MI between the two groups: 12.6% in the placebo group and 8.8% in the clopidogrel group experienced primary outcomes (P = 0.002). PCI-CURE investigators concluded that compared to placebo, patients with non-ST elevation MI treated with clopidogrel plus aspirin and PCI had a reduced risk of cardiovascular death and MI by about a third. Long-term therapy was also associated with a lower rate of cardiovascular death, MI, or the need for revascularization (P = 0.03). There was no significant difference in major, but not life-threatening, bleeding with clopidogrel. The results of the PCI-CURE study supported the use of combination antiplatelet therapy with clopidogrel plus aspirin in patients undergoing PCI with ACS and non-ST-segment elevation MI. It was further suggested that long-term combination therapy is beneficial in reducing major cardiovascular events. 2.2.6. The MATCH Study

In the MATCH trial, 7,599 high-risk patients with cerebrovascular disease were treated with clopidogrel or clopidogrel plus aspirin and assessed for vascular events (43). Patients admitted into this trial were stable and receiving clopidogrel (75 mg/day), and were randomized to either placebo or low-dose aspirin (75 mg/day). Patients had to have experienced an ischemic stroke or transient ischemic attack (TIA) within the past 3 months, and they had to have one or more of the following four risk factors: previous MI, angina pectoris, diabetes mellitus, and symptoms of PAD within the past 3 years. The primary end point for the MATCH trial was a composite of ischemic stroke, MI, vascular death, and rehospitalization for an acute ischemic event (angina pectoris, worsening PAD symptoms, or TIA). Secondary outcomes included death from any type of stroke. The MATCH trial failed to show any significant difference in risk reduction between the clopidogrel plus aspirin group versus the clopidogrel plus placebo group. There was no statistical significance in any of the primary end points between the two arms that indicated a reduction in rates of ischemic stroke, MI, vascular death, or rehospitalization for any acute ischemic event. In addition, there was a significantly higher rate of major bleeding in the combination therapy group (P< 0.0001). The risk was

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1.36% higher in the combination arm as compared to the clopidogrel monotherapy arm. Thus, there was no significant reduction in vascular events by adding aspirin to clopidogrel for high-risk patients with ischemic stroke or TIA. 2.2.7. The CARESS Study

In the CARESS (Clopidogrel and Aspirin for Reduction of Emboli in Symptomatic Carotid Stenosis) trial, Markus et al. (44) evaluated combination therapy in cerebrovascular risk patients. All patients were recently symptomatic, with ≥50% carotid stenosis with micro-embolic signals (MES), as detected by transcranial Doppler ultrasound. Patients were randomized to clopidogrel (loading dose of 300 mg on day 1 followed by 75 mg/day) or placebo. All patients also received aspirin 75 mg/day for the duration of the study. Patients were all >18 years of age, had >50% carotid stenosis, and had experienced ipsilateral carotid territory TIA or stroke within the preceding 3 months. The concomitant use of anticoagulants, thrombolytic agents, analgesics (other than acetaminophen or opioids), and any additional antiplatelet agents was prohibited during the course of the study. The CARESS trial assessed the proportion of patients who were MES-positive on a 1-h recording conducted on day 7 of the trial. A reduction in the MES value would correspond to decreased markers of platelet and thrombus emboli in the ipsilateral middle cerebral artery, leading to a lower risk of recurrent strokes in this patient population. Safety end points were recorded as any adverse or cerebrovascular events, such as TIA, ischemic stroke, or cerebral hemorrhage. Bleeding events were divided into three categories: lifethreatening, major, or minor bleeding. Of the 107 randomized patients who had an MES value >1, 43.8% of patients in the combination group versus 72.7% in the placebo group had a positive MES reading on day 7, favoring combination therapy. There was a relative risk reduction of 39.8% for those patients who received clopidogrel plus aspirin versus aspirin alone. This was statistically significant (P = 0.0046), with no further increases in bleeding events. The CARESS study concluded that in “actively embolizing” patients with recently symptomatic carotid stenosis (>50%), the combination of clopidogrel plus aspirin therapy is more effective than aspirin alone in reducing asymptomatic embolization, with a relative risk reduction of 40% in 7 days. Active treatment response was seen with a similar magnitude of effect on day 2. These findings were in contrast to the results of the MATCH study; however, there are some differences to be noted between the two patient populations. The MATCH study data were based on all types of ischemic stroke, including small-vessel disease, which has the lowest risk of early recurrent stroke because it is not a process caused by embolism from an atherosclerotic plaque (45). This may be one of the reasons why interpretation of the

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MATCH trial data at 18 months is not consistent with that of the CARESS trial after 7 days. The MATCH trial also included patients who were experiencing events several weeks after their acute phase. This group of patients is at the highest risk for recurrent stroke. In contrast, the CARESS study was consistent with reducing recurrent stroke in patients with large-vessel atherosclerotic stroke in the acute phase. The difference in the power of these two studies should also be noted. The CARESS study analyzed 107 patients versus 7,276 patients analyzed in the MATCH trial. Upon direct comparison, the MATCH trial had a greater power value than the CARESS study, which could also influence the observed outcomes. 2.2.8. Summary: Combination Aspirin and Clopidogrel Therapy

There is considerable clinical evidence supporting the use of combination antiplatelet therapy with clopidogrel and aspirin. The CLARITY-TIMI and the COMMIT trials both studied patients suffering from acute MI with ST-segment elevations or newly developed left-bundle branch block (39, 40). These two trials demonstrated greater efficacy when patients were given clopidogrel plus aspirin versus aspirin alone. Death, reinfarction, and stroke were all reduced with the combination of antiplatelets, with no significant difference in major bleeding compared to aspirin alone. The CURE trial was conducted to investigate the reduction of risks in patients undergoing vascular interventions. Again, patients who received clopidogrel and aspirin before surgery experienced a significant reduction in the primary end points. These results were supported by the PCI-CURE study (42). Asymptomatic patients who were considered to be at high risk for atherothrombotic events did not benefit from combination antiplatelet therapy in the CHARISMA study (41). Clopidogrel plus aspirin is not recommended for the prevention of atherothrombotic events in stable patients who have multiple risk factors alone. The MATCH and CARESS trials raised much controversy due to their conflicting results. The CARESS trial observed patients who experienced ipsilateral carotid territory TIA/stroke only (43–45). Furthermore, observations in the MATCH trial were recorded up to 18 months, whereas the CARESS results were recorded after 7 days. One issue that deserves further discussion is the duration of therapy. There is conflicting evidence from the MATCH and CARESS trials as to the optimal duration of antiplatelet therapy in cerebrovascular disease. For coronary artery disease, the CHARISMA trial failed to show a benefit of long-term clopidogrel in the overall trial population, although the 80% of patients with clinically evident atherothrombosis experienced a modest reduction of the primary endpoint, and emerging data with drug-eluting stents suggest that dual antiplatelet therapy may

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be required even beyond 1 year. Clearly, additional studies are needed to evaluate optimal antiplatelet therapy combinations and duration of therapies to permit maximal benefit with the minimum of harm in patients with cardiovascular disease.

3. Expert Opinion and Future Directions

Platelet aggregation plays a key role in the pathogenesis of coronary thrombosis, and pharmacologic inhibition of platelet function forms the cornerstone of treatment for ACS. Cardiovascular treatment with clopidogrel versus aspirin has been shown to be beneficial, but with adequate precautions (46). When clopidogrel and aspirin were each studied in monotherapy, clopidogrel proved to be superior to aspirin in reducing the risk of ischemic events (47). When the two were combined in the first trial to show effectiveness versus aspirin monotherapy, the combination was associated with a high risk of bleeding (46). Only when investigators began to observe carefully specific patient populations did combination therapy begin to demonstrate promising results (e.g., the PCI-CURE study). Clopidogrel has been shown to be beneficial in the initial treatment and secondary prevention of ACS. Compared with aspirin alone, dual antiplatelet therapy with aspirin plus clopidogrel reduces the risk of vessel thrombosis and recurrent ischemic events in patients undergoing PCI and is a useful adjunct to coronary artery stenting. In CABG surgery, combination aspirin and clopidogrel therapy initiated immediately postoperatively improves bypass graft patency. The search for ADP receptor antagonists with a rapid onset and short half-life is ongoing. Such agents would allow for urgent surgical procedures and would overcome the current limitations of clopidogrel. Furthermore, antiplatelet agents that work via collagen receptors, serotonin receptors, or thrombin receptors may have additional value and the ability to complement current antiplatelet therapy. Several rapid-onset and rapid-offset reversible ADP antagonists are currently in clinical development (35, 48). AZD-6140 is a reversible oral P2Y12 receptor antagonist that has been studied in ACS patients in comparison to clopidogrel in the DISPERSE-2 (Dose Confirmation Study Assessing Antiplatelet Effects of AZD6140 versus Clopidogrel in Non-ST-Segment Elevation MI) study. AZD-6140 exhibited greater mean inhibition of platelet aggregation than a standard regimen of clopidogrel in ACS patients. In addition, AZD-6140 further suppressed platelet aggregation in clopidogrel-pretreated patients (48).

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One could extrapolate that reversible ADP receptor antagonists would have greater clinical benefits based on ex vivo studies showing that their ability to inhibit platelet aggregation is superior to other drugs such as clopidogrel. However, as demonstrated by the case of oral GPIIb/IIIa inhibitors, promising laboratory results do not necessarily translate into successful outcomes in the clinical setting. Despite consistent evidence of substantial inhibition of platelet aggregation by oral GPIIb/IIIa inhibitors, this class of drugs provided no clinical benefit in phase-III trials and, in fact, was harmful. The connection between ex vivo inhibition of platelet aggregation and clinical benefit of platelet P2Y12 antagonists is also not straightforward. A link between inflammatory status and clinical benefit from antiplatelet agents continues to emerge and highlights the fact that biomarkers beyond ex vivo platelet aggregation might better predict the clinical benefit of antiplatelet agents that reduce platelet activation. Ongoing trials hold promise for determining the appropriate targets for maximizing antiplatelet efficacy, but the current lack of a proven ex vivo assay that correlates with clinical outcomes hampers clinical investigation, particularly in light of the expense involved in conducting these mega trials. ADP receptor blockers require 3–7 days to reach maximum inhibition of platelet aggregation. When investigators began to assess the use of a loading dose with clopidogrel, therapeutic results occurred more rapidly (33, 44). There was a rapid onset of platelet aggregate inhibition, with an antithrombotic effect observed within 90 min. The optimal type of antiplatelet therapy for patients who will undergo surgery would be one with a rapid onset and a relatively short elimination half-life, allowing for a once- or twice-daily regimen. A significant impediment to the development and clinical application of antiplatelet therapies is the prohibitive cost of clinical trials and the potential risk involved in attaining clinical superiority and safety over existing regimens. The recruitment and study of a large number of patients for a prolonged period of time may be necessary to demonstrate a modest clinical benefit. Thus, the prohibitive cost of clinical trials demonstrating significant differences to current antiplatelet therapies might limit progress in advancing new antiplatelet targets.

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Mousa platelet glycoprotein IIb/IIIa receptor in high-risk coronary angioplasty. N Engl J Med 330, 956–61. Fitchett, D.H., Langer, A., Armstrong, P.W., Tan, M., Mendelsohn, A., and Goodman, S.G. (2006) Randomized evaluation of the efficacy of enoxaparin versus unfractionated heparin in high-risk patients with non-STsegment elevation acute coronary syndromes receiving the glycoprotein IIb/IIIa inhibitor eptifibatide. Long-term results of the Integrilin and Enoxaparin Randomized Assessment of Acute Coronary Syndrome Treatment (INTERACT) trial Am Heart J 151, 373–9. Hayes, R., Chesebro, J.H., Fuster, V., Dangas, G., Fallon, J.T., Sharma, S.K., Coller, B.S., Badimon, L., Marmur, J.D., and Badimon, J.J. (2000) Antithrombotic effects of abciximab Am J Cardiol 85, 1167–72. Januzzi, J.L., Jr., Snapinn, S.M., DiBattiste, P.M., Jang, I.K., and Theroux, P. (2002) Benefits and safety of tirofiban among acute coronary syndrome patients with mild to moderate renal insufficiency: results from the Platelet Receptor Inhibition in Ischemic Syndrome Management in Patients Limited by Unstable Signs and Symptoms (PRISMPLUS) trial Circulation 105, 2361–6. Lincoff, A.M., Califf, R.M., Van de Werf, F., Willerson, J.T., White, H.D., Armstrong, P.W., Guetta, V., Gibler, W.B., Hochman, J.S., Bode, C., Vahanian, A., Steg, P.G., Ardissino, D., Savonitto, S., Bar, F., Sadowski, Z., Betriu, A., Booth, J.E., Wolski, K., Waller, M., and Topol, E.J. (2002) Mortality at 1 year with combination platelet glycoprotein IIb/IIIa inhibition and reduceddose fibrinolytic therapy vs conventional \hbox{fibrinolytic} therapy for acute myocardial infarction: GUSTO V randomized trial JAMA 288, 2130–5. Neumann, F.J., Kastrati, A., Schmitt, C., Blasini, R., Hadamitzky, M., Mehilli, J., Gawaz, M., Schleef, M., Seyfarth, M., Dirschinger, J., and Schomig, A. (2000) Effect of glycoprotein IIb/IIIa receptor blockade with abciximab on clinical and angiographic restenosis rate after the placement of coronary stents following acute myocardial infarction J Am Coll Cardiol 35, 915–21. Anderson, J.L., Adams, C.D., Antman, E.M., Bridges, C.R., Califf, R.M., Casey, D.E., Jr., Chavey, W.E., 2nd, Fesmire, F.M., Hochman, J.S., Levin, T.N., Lincoff, A.M., Peterson, E.D., Theroux, P., Wenger, N.K., Wright, R.S., Smith, S.C., Jr., Jacobs, A.K., Halperin, J.L., Hunt, S.A., Krumholz, H.M.,

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Kushner, F.G., Lytle, B.W., Nishimura, R., Ornato, J.P., Page, R.L., and Riegel, B. (2007) ACC/AHA 2007 guidelines for the management of patients with unstable angina/non-ST-elevation myocardial infarction: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Revise the 2002 Guidelines for the Management of Patients With Unstable Angina/Non-ST-Elevation Myocardial Infarction) developed in collaboration with the American College of Emergency Physicians, the Society for Cardiovascular Angiography and Interventions, and the Society of Thoracic Surgeons endorsed by the American Association of Cardiovascular and Pulmonary Rehabilitation and the Society for Academic Emergency Medicine J Am Coll Cardiol 50, e1–157. Cadroy, Y., Bossavy, J.P., Thalamas, C., Sagnard, L., Sakariassen, K., and Boneu, B. (2000) Early potent antithrombotic effect with combined aspirin and a loading dose of clopidogrel on experimental arterial thrombogenesis in humans Circulation 101, 2823–8. Coma-Canella, I., Velasco, A., and Castano, S. (2005) Prevalence of aspirin resistance measured by PFA-100 Int J Cardiol 101, 71–6. Wiviott, S.D., Braunwald, E., McCabe, C.H., Montalescot, G., Ruzyllo, W., Gottlieb, S., Neumann, F.J., Ardissino, D., De Servi, S., Murphy, S.A., Riesmeyer, J., Weerakkody, G., Gibson, C.M., and Antman, E.M. (2007) Prasugrel versus clopidogrel in patients with acute coronary syndromes N Eng J Med 357, 2001–15. Angiolillo, D.J., Shoemaker, S.B., Desai, B., Yuan, H., Charlton, R.K., Bernardo, E., Zenni, M.M., Guzman, L.A., Bass, T.A., and Costa, M.A. (2007) Randomized comparison of a high clopidogrel maintenance dose in patients with diabetes mellitus and coronary artery disease: results of the Optimizing Antiplatelet Therapy in Diabetes Mellitus (OPTIMUS) study Circulation 115, 708–16. Spertus, J.A., Kettelkamp, R., Vance, C., Decker, C., Jones, P.G., Rumsfeld, J.S., Messenger, J.C., Khanal, S., Peterson, E.D., Bach, R.G., Krumholz, H.M., and Cohen, D.J. (2006) Prevalence, predictors, and outcomes of premature discontinuation of thienopyridine therapy after drug-eluting stent placement: results from the PREMIER registry Circulation 113, 2803–9.

Antiplatelet Therapies: Drug Interactions in the Management of Vascular Disorders 38. Smith, S.C., Jr., Feldman, T.E., Hirshfeld, J.W., Jr., Jacobs, A.K., Kern, M.J., King, S.B., 3rd, Morrison, D.A., O Neill, W.W., Schaff, H.V., Whitlow, P.L., Williams, D.O., Antman, E.M., Adams, C.D., Anderson, J.L., Faxon, D.P., Fuster, V., Halperin, J.L., Hiratzka, L.F., Hunt, S.A., Nishimura, R., Ornato, J.P., Page, R.L., and Riegel, B. (2006) ACC/AHA/SCAI 2005 guideline update for percutaneous coronary intervention: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (ACC/AHA/SCAI Writing Committee to Update the 2001 Guidelines for Percutaneous Coronary Intervention) J Am Coll Cardiol 47, e1–121. 39. Sabatine, M.S., Cannon, C.P., Gibson, C.M., Lopez-Sendon, J.L., Montalescot, G., Theroux, P., Claeys, M.J., Cools, F., Hill, K.A., Skene, A.M., McCabe, C.H., and Braunwald, E. (2005) Addition of clopidogrel to aspirin and fibrinolytic therapy for myocardial infarction with ST-segment elevation N Eng J Med 352, 1179–89. 40. Chen, Z.M., Jiang, L.X., Chen, Y.P., Xie, J.X., Pan, H.C., Peto, R., Collins, R., and Liu, L.S. (2005) Addition of clopidogrel to aspirin in 45,852 patients with acute myocardial infarction: randomised placebocontrolled trial Lancet 366, 1607–21. 41. Bhatt, D.L., Fox, K.A., Hacke, W., Berger, P.B., Black, H.R., Boden, W.E., Cacoub, P., Cohen, E.A., Creager, M.A., Easton, J.D., Flather, M.D., Haffner, S.M., Hamm, C.W., Hankey, G.J., Johnston, S.C., Mak, K.H., Mas, J.L., Montalescot, G., Pearson, T.A., Steg, P.G., Steinhubl, S.R., Weber, M.A., Brennan, D.M., Fabry-Ribaudo, L., Booth, J., and Topol, E.J. (2006) Clopidogrel and aspirin versus aspirin alone for the prevention of atherothrombotic events N Eng J Med 354, 1706–17. 42. Mehta, S.R., Yusuf, S., Peters, R.J., Bertrand, M.E., Lewis, B.S., Natarajan, M.K., Malmberg, K., Rupprecht, H., Zhao, F., Chrolavi-

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cius, S., Copland, I., and Fox, K.A. (2001) Effects of pretreatment with clopidogrel and aspirin followed by long-term therapy in patients undergoing percutaneous coronary intervention: the PCI-CURE study Lancet 358, 527–33. Diener, H.C., Bogousslavsky, J., Brass, L.M., Cimminiello, C., Csiba, L., Kaste, M., Leys, D., Matias-Guiu, J., and Rupprecht, H.J. (2004) Aspirin and clopidogrel compared with clopidogrel alone after recent ischaemic stroke or transient ischaemic attack in high-risk patients (MATCH): randomised, double-blind, placebo-controlled trial Lancet 364, 331–7. Markus, H.S., Droste, D.W., Kaps, M., Larrue, V., Lees, K.R., Siebler, M., and Ringelstein, E.B. (2005) Dual antiplatelet therapy with clopidogrel and aspirin in symptomatic carotid stenosis evaluated using doppler embolic signal detection: the Clopidogrel and Aspirin for Reduction of Emboli in Symptomatic Carotid Stenosis (CARESS) trial Circulation 111, 2233–40. Rothwell, P.M. (2004) Lessons from MATCH for future randomised trials in secondary prevention of stroke Lancet 364, 305–7. Hirsh, J., and Bhatt, D.L. (2004) Comparative benefits of clopidogrel and aspirin in high-risk patient populations: lessons from the CAPRIE and CURE studies Arch Intern Med 164, 2106–10. CAPRIE Steering Committee (1996) A randomised, blinded, trial of clopidogrel versus aspirin in patients at risk of ischaemic events (CAPRIE). Lancet 348, 1329–39. Storey, R.F., Husted, S., Harrington, R.A., Heptinstall, S., Wilcox, R.G., Peters, G., Wickens, M., Emanuelsson, H., Gurbel, P., Grande, P., and Cannon, C.P. (2007) Inhibition of platelet aggregation by AZD6140, a reversible oral P2Y12 receptor antagonist, compared with clopidogrel in patients with acute coronary syndromes J Am Coll Cardiol 50, 1852–6.

Chapter 8 Prasugrel: A Novel Platelet ADP P2Y12 Receptor Antagonist Shaker A. Mousa, Walter P. Jeske, and Jawed Fareed Abstract Novel adenosine diphosphate (ADP) P2Y12 antagonists such as prasugrel, ticagrelor, cangrelor, and elinogrel are in various phases of clinical development. These ADP P2Y12 antagonists have advantages over clopidogrel ranging from faster onset to greater and less variable inhibition of platelet function. Novel ADP P2Y12 antagonists are under investigation to determine whether their use can result in improved antiplatelet activity, faster onset of action, and/or greater antithrombotic effects than clopidogrel without an unacceptable increase in hemorrhagic or other side effects. Prasugrel (CS-747; LY640315), a novel third-generation oral thienopyridine, is a specific, irreversible antagonist of the platelet ADP P2Y12 receptor. Pre-clinical and early phase clinical studies have shown prasugrel to be characterized by more potent antiplatelet effects, lower inter-individual variability in platelet response, and faster onset of activity compared to clopidogrel. Recent findings from large-scale phase-III testing show prasugrel to be more efficacious in preventing ischemic events in acute coronary syndrome patients undergoing percutaneous coronary intervention (PCI); however, this is achieved at the expense of an increased risk of bleeding. Prasugrel provides more rapid and consistent platelet inhibition than clopidogrel. Key words: Antiplatelet, acute coronary syndrome, antithrombotic, percutaneous coronary intervention, platelets, thienopyridines, thrombosis, antiplatelet combinations.

1. Introduction Platelets are the principle effectors of cellular hemostasis and key mediators in the pathogenesis of thrombosis. A variety of membrane receptors determine platelet reactivity with numerous agonists and adhesive proteins, and, therefore, represent key targets for the development of antiplatelet drug therapies. In this regard, several rapid-onset and rapid-offset reversible ADP antagonists are in clinical development, including reversible oral and rapid acting intravenous (1, 2) P2Y12 receptor antagonists (Table 8.1). S.A. Mousa (ed.), Anticoagulants, Antiplatelets, and Thrombolytics, Methods in Molecular Biology 663, DOI 10.1007/978-1-60761-803-4_8, © Springer Science+Business Media, LLC 2003, 2010

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Table 8.1 Promising new antiplatelet P2Y12 inhibitor therapies in development Agent

Potential advantages (clopidogrel, ticlopidine)

over

current

agents

Prasugrel

Faster onset, greater antiplatelet effect, less variable response (FDA approved)

AZD6140

Reversible, faster onset and offset, greater antiplatelet effect

Cangrelor (IV)

Reversible, shorter half-life, faster onset, greater antiplatelet effect, less variable response

Novel inhibitors of platelet adhesion in early development target the von Willebrand (vWF)-GPIb/IX and collagen/GPVI interactions. Since platelet aggregation also plays such a critical role in the pathogenesis of arterial thrombosis, more potent agents that interfere with platelet aggregation via other pathways (e.g., the thrombin receptor) are also under clinical investigation (3) The major limitation to treatment with multiple antiplatelet agents is the increased bleeding risk associated with the enhanced antiplatelet effect. This is exemplified by the clinical conundrum in patients with acute coronary syndrome (ACS) who may need to undergo coronary artery bypass graft (CABG) surgery. Aspirin and clopidogrel irreversibly inhibit platelet function, with maximal antiplatelet effect occurring after 3–5 days of treatment. The increased risk of procedural bleeding arising from dual aspirin and clopidogrel administration immediately prior to CABG surgery raises the question of whether clopidogrel should be routinely given to patients presenting with ACS. In light of the current recommendation to discontinue clopidogrel at least 5 days prior to elective CABG surgery, the emergency physician is likely to avoid clopidogrel in anticipation that the patient may require urgent cardiac surgery. Delaying clopidogrel therapy until coronary revascularization has been performed would, however, deprive patients of the early clinical benefits of the drug. These limitations might be solved with the availability of rapid-onset and rapid-offset ADP antagonists. Furthermore, it is becoming clear that there is variability in individual responses to clopidogrel, with reported rates of inadequate antiplatelet response ranging between 4 and 30% (4). Reasons for this include genetic variables (polymorphisms of the P2Y12 receptor or CYP3A4 pathway), up-regulation of alternative pathways of platelet activation, and greater baseline pre-treatment platelet reactivity, as well as extrinsic mechanisms such as patient non-compliance and drug–drug interactions involving CYP3A4 (4). Hence, a clinical need exists for superior antiplatelet agents.

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2. Prasugrel 2.1. Pharmacokinetics

Prasugrel is a pro-drug requiring activation by the hepatic CYP system. As it is rapidly absorbed, prasugrel has a faster onset of action compared to clopidogrel, with peak concentrations of active metabolite seen at 30 min (5). Prasugrel’s active metabolite is predominantly renally excreted (approximately 70%) and has a mean elimination half-life of 3.7 h. Important metabolic differences between clopidogrel and prasugrel result in a higher concentration of active metabolite with prasugrel. Approximately 85% of clopidogrel is hydrolyzed by esterases to an inactive carboxylic acid derivative, leaving 15% of the pro-drug to be metabolized in a two-step CYP process into active metabolite. Prasugrel, by contrast, is rapidly hydrolyzed by carboxyesterases and then metabolized in a single, CYP-dependent step that uses primarily isotypes CYP3A4 and CPY2B6 (6), which translates into improved inhibition of platelet aggregation on a mg/kg basis compared with clopidogrel. Current evidence from phase-I studies demonstrates that prasugrel, when compared to clopidogrel, provides greater inhibition of platelet aggregation with more rapid onset and less nonresponsiveness, most likely due to more efficient generation of the prasugrel active metabolite. In one study, 68 healthy subjects not taking aspirin received either a 300-mg loading dose of clopidogrel (then 75 mg daily) or a 60-mg loading dose of prasugrel (then 10 mg daily), followed by the alternate therapy after a 2week washout (7). The peak inhibitory effect on platelet aggregation was greater with prasugrel (mean inhibition, 79% versus 35%, P<0.001), while onset of antiplatelet activity was more rapid (maximal inhibitory effect achieved in 60 min with prasugrel versus 4–6 h with clopidogrel). Drug resistance in this study was defined as <20% inhibition of platelet aggregation (IPA) at 24 h and was seen in 42% of the clopidogrel-treated patients and none of the prasugrel-treated patients (7).

2.2. Therapeutic Review

A multiple oral dose phase-I study involving 30 patients demonstrated benefit in terms of the maximum level of platelet inhibition for all prasugrel doses (5, 10, or 20 mg) over clopidogrel 75 mg (P<0.001) (8). A 60-mg loading dose was been shown to provide faster onset of action and greater IPA than either a 300- or 600-mg loading dose of clopidogrel (9). Maintenance doses of 10 and 15 mg daily of prasugrel were superior to clopidogrel 75 mg daily in a phase-Ib study, with greater platelet inhibition (61 versus 68 versus 30%, P<0.0001) and less incidence of non-responsiveness (10). Such results supported proceeding to phase-II testing, which has demonstrated an acceptable safety

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profile for prasugrel as compared to clopidogrel. In particular, in the JUMBO-TIMI 26 trial, which compared prasugrel to clopidogrel use in 900 patients undergoing elective or urgent percutaneous coronary intervention (PCI), no statistical difference in TIMI major and minor bleeding was noted (1.7 versus 1.2%, P = 0.59). The composite endpoint of 30-day major adverse cardiac events trended lower in the prasugrel-treated patients (7.2 versus 9.4%, P=0.26), but was not statistically significant (11). The PRINCIPLE-TIMI 44 study was the first to compare prasugrel 10 mg daily (after a 60-mg loading dose) to high dose clopidogrel (150 mg daily after a 600-mg loading dose) in patients undergoing catheterization for planned PCI (12). Prasugrel-treated patients had more consistent levels of platelet inhibition with lower inter-individual variability. IPA at 6 h was significantly higher in the prasugrel group (74 versus 31%, P<0.0001), and the effect was maintained at 14 days. No TIMI major bleeds occurred in this study of 201 patients, while TIMI minor bleeds occurred in 2 patients (2%) in the prasugrel group versus none in the clopidogrel group. Data from the phase III TRITON-TIMI 38 (Trial to Assess Improvement in Therapeutic Outcomes by Optimizing Platelet Inhibition with Prasugrel-TIMI 38) trial demonstrated improved clinical outcomes with prasugrel as compared to clopidogrel. TRITON-TIMI 38 was a double-blind, randomized controlled trial comparing prasugrel with clopidogrel in 13,608 patients with moderate- to high-risk ACS (26% STEMI, 74% unstable angina or NSTEMI) who underwent PCI (13). Patients were randomized to treatment with either prasugrel (60-mg loading dose then 10 mg/day maintenance) or clopidogrel (300-mg loading dose then 75 mg /day). The median duration of therapy was 14.5 months. The primary endpoint, a composite of cardiovascular death, nonfatal myocardial infarction (MI) or nonfatal stroke, occurred in 9.9% of the prasugrel group versus 12.1% of clopidogrel-treated patients (hazard ratio 0.81, 95% confidence interval, 0.73–0.90; P<0.001), supporting the superior efficacy of prasugrel. This benefit persisted throughout the follow-up period, suggesting a continued benefit of greater platelet inhibition during the maintenance phase of therapy, and was evident in both subgroups of unstable angina/NSTEMI and STEMI. The prasugrel group also showed a significant reduction in the secondary endpoints of death from cardiovascular causes, nonfatal infarction, or urgent target vessel revascularization at 30 days (HR 0.78, P=0.02) and at 90 days (HR 0.79, P<0.001). Significant reductions in the prasugrel group were seen in the rates of MI (7.3% versus 9.4%, HR 0.76, P<0.001), urgent target vessel revascularization (2.5% versus 3.7%, HR 0.66, P<0.001), and stent thrombosis (1.1% versus 2.4%, HR 0.48, P<0.001). Notably, the reductions in stent thrombosis were irrespective of stent type or

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timing (early, <30 days and late, >30 days) (14). It should be noted that stent use was non-randomized. The major drawback for prasugrel identified in this trial was an increased risk of major bleeding, an important issue given its association with mortality. Major bleeding was observed in 2.4% of the patients receiving prasugrel and 1.8% of those receiving clopidogrel (HR 1.32, 95% 1.03–1.68; P=0.03). The rate of lifethreatening bleeding was greater in the prasugrel group (1.4 versus 0.9%, P=0.01), including nonfatal (1.1 versus 0.9%, P=0.01) and fatal bleeding (0.4 versus 0.1%, P=0.002). TIMI minor bleeds were more frequent in the prasugrel group as was major non-CABG bleeding (14). Despite this increase in bleeding, the pre-specified net clinical benefit analysis, which was defined as the composite of efficacy (death from any cause, nonfatal MI, nonfatal stroke) and bleeding endpoints (TIMI major hemorrhage), still favored prasugrel (12.2% in the prasugrel group versus 13.9% in the clopidogrel group; HR 0.87, 95% CI 0.79–0.95: P=0.004). This net clinical benefit for prasugrel existed both early and late in the trial. The use of prasugrel in place of clopidogrel essentially prevents 23 acute myocardial infarcts per 1,000 patients treated at the expense of five additional major bleeds (per 1,000 patients). Post hoc analysis has identified three subgroups with less net clinical benefit or net harm with the use of prasugrel, driven by excess bleeding risk: previous cerebrovascular events, age >75 or weight <60 kg (15). Patients with a history of stroke or transient ischemic attack had net harm (HR 1.54, 95% CI 1.02– 2.32; P=0.04). This group had no evidence of clinical benefit with prasugrel when compared to clopidogrel, as evaluated by the primary efficacy endpoint, and had a greater rate of TIMI major bleeding (5.0 versus 2.9%, P=0.06), including intracranial hemorrhage (2.3 versus 0%, P=0.02). Patients 75 years of age or older had no net benefit (HR 0.99, 95% CI 0.81−1.21; P=0.92), nor did patients weighing <60 kg (HR 1.03, 95% CI 0.69−1.53; P=0.89). Interestingly, two other subgroups appeared to derive significant net benefit from prasugrel as compared to clopidogrel: STEMI patients and those with diabetes. In the STEMI cohort of 3,500 patients, there was a 21% relative risk reduction in the primary endpoint of cardiovascular death, MI, or stroke with prasugrel (10.0 versus 12.4%, HR 0.79, P=0.02) (16, 17). This endpoint reduction was driven by less recurrent MI and stent thrombosis with no difference in mortality and no increase in major bleeding. A greater reduction in ischemic events and MI with no increase in bleeding compared to clopidogrel was clearly evident in patients with diabetes (30% risk reduction). The reason for this lack of bleeding is unclear but may represent possible protection from bleeding in STEMI and diabetic patients due to increased platelet activation. Another

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phase-III trial in development is the TRILOGY ACS study (Targeted Platelet Inhibition to Clarify the Optimal Strategy to Medically Manage Acute Coronary Syndromes) (18). This doubleblind, randomized controlled trial will evaluate the efficacy and safety of prasugrel versus clopidogrel in ACS patients who are medically managed and not planned for revascularization (18). 2.3. Prasugrel in Renal Impairment and End-Stage Renal Disease

Pharmacokinetic (PK) and pharmacodynamic (PD) responses to prasugrel were compared in three studies of healthy subjects versus those with moderate- or end-stage renal impairment. The data showed no difference in PK or PD responses between healthy subjects and subjects with moderate renal impairment (19). PK and PD studies with prasugrel in moderate hepatic impairment also showed little to no effect on PK or platelet aggregation relative to healthy controls, suggesting that a dose adjustment would not be required in patients with moderate liver diseases (20).

3. Conclusion Prasugrel is a third-generation thienopyridine that selectively inhibits the platelet P2Y12 receptor. It leads to platelet inhibition more rapidly, more potently, and with less inter-individual variability as compared to clopidogrel. Such pharmacodynamic properties reflect pharmacokinetic differences, namely a more efficient metabolism of prasugrel into its active metabolite compared to clopidogrel. Phase-III testing in high-risk patients undergoing PCI showed long-term prasugrel use translates into improved clinical outcomes compared to clopidogrel. Increased risk of TIMI major bleeding was demonstrated but the net clinical outcome still favors prasugrel over clopidogrel. Clearly, minimizing bleeding risk, perhaps by dose modifications in specific populations, will maximize the clinical benefit of prasugrel. Further clinical studies will evaluate prasugrel use in other clinical scenarios and hopefully better define the patient populations who will best benefit from this novel antiplatelet agent. The bar has been raised for therapeutic regimens employing aspirin, clopidogrel, and their combination. Antiplatelet therapies with enhanced efficacy and parallel safety profiles are desired, but such agents may be mutually exclusive and difficult to attain. Such was the case in the TRITON-TIMI-38 study (21), in which enhanced antiplatelet therapy and a significant reduction in ischemic events with prasugrel were accompanied by a significant risk of fatal and life-threatening bleeding in comparison to clopidogrel. Newer antiplatelet agents that target different mechanisms are also being developed and studied in addition to

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aspirin and clopidogrel (22). The major challenges faced by these newer agents include not only the difficulty in proving incremental meaningful clinical benefit, but doing so with a minimal increase in bleeding risk against a background of contemporary therapies which themselves continue to evolve.

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8. Jakubowski, J.A., Matsushima, N., Asai, F., et al. (2007) A multiple dose study of prasugrel (CS- 747), a novel thienopyridine P2Y12 inhibitor, compared with clopidogrel in healthy humans Br J Clin Pharmacol 63, 421–30. 9. Haynes, R.B., Sandler, R.S., Larson, E.B., et al. (1992) A critical appraisal of ticlopidine, a new antiplatelet agent: effectiveness and clinical indications for prophylaxis of atherosclerotic events Arch Intern Med 152, 1376–80. 10. Jernberg, T., Payne, C.D., Winters, K.J., et al. (2006) Prasugrel achieves greater inhibition of platelet aggregation and a lower rate of non-responders compared with clopidogrel in aspirin-treated patients with stable coronary artery disease Eur Heart J 27, 1166–73. 11. Wiviott, S.D., Antman, E.M., Winters, K.J., et al. (2005) Randomized comparison of prasugrel (CS-747, LY640315), a novel thienopyridine P2Y12 antagonist, with clopidogrel in percutaneous coronary intervention: results of the Joint Utilization of Medications to Block Platelets Optimally (JUMBO)-TIMI 26 Trial Circulation 111, 3366–73. 12. Wiviott, S.D., Trenk, D., Frelinger, A.L., et al. (2007) Prasugrel compared with high loading- and maintenance-dose clopidogrel in patients with planned percutaneous coronary intervention: the prasugrel in comparison to clopidogrel for inhibition of platelet activation and aggregation – thrombolysis in myocardial infarction 44 trial Circulation 116, 2923–32. 13. Wiviott, S.D., Braunwald, E., McCabe, C.H., et al. (2007) Prasugrel versus clopidogrel in patients with acute coronary syndromes N Engl J Med 357, 2001–15. 14. Wiviott, S.D., Braunwald, E., McCabe, C.H., et al. (2008) Intensive oral antiplatelet therapy for reduction of ischaemic events including stent thrombosis in patients with acute coronary syndromes treated with percutaneous coronary intervention and

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16. 17. 18.

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Mousa, Jeske, and Fareed stenting in the TRITONTIMI 38 trial: a subanalysis of a randomized trial Lancet 371, 1353–63. Antman, E.M., Wiviott, S.D., Murphy, S.A., et al. (2008) Early and late benefits of prasugrel in patients with acute coronary syndromes undergoing percutaneous coronary intervention A TRITON-TIMI 38 (Trial to Assess Improvement in Therapeutic Outcomes by Optimizing Platelet Inhibition with Prasugrel – Thrombolysis in Myocardial Infarction) J Am Coll Cardiol 51, 2028–33. Floyd, J., and Wolfe, S. (2009) Prasugrel STEMI subgroup analysis Lancet 373, 1845–6. Webster, M.W., and Gladding, P. (2009) Prasugrel STEMI subgroup analysis Lancet 373, 1846–8. Spinler, S.A., and Rees, C. (2009) Review of prasugrel for the secondary prevention of atherothrombosis J Manag Care Pharm 15, 383–95. Small, D.S., Wrishko, R.E., Ernest, C.S. 2nd, Ni, L., Winters, K.J., Farid, N.A., Li, Y.G., Brandt, J.T., Salazar, D.E., Borel, A.G., Kles,

K.A., and Payne, C.D. (2009) Prasugrel pharmacokinetics and pharmacodynamics in subjects with moderate renal impairment and end-stage renal disease J Clin Pharm Ther 34, 585–94. 20. Small, D.S., Farid, N.A., Li, Y.G., Ernest, C.S. 2nd, Winters, K.J., Salazar, D.E., and Payne, C.D. (2009) Pharmacokinetics and pharmacodynamic of prasugrel in subjects with moderate liver disease J Clin Pharm Ther 34, 575–83. 21. Montalescot, G., Wiviott, S.D., Braunwald, E., Murphy, S.A., Gibson, C.M., McCabe, C.H., Antman, E.M., for the TRITONTIMI 38 Investigators. (2009) Prasugrel compared with clopidogrel in patients undergoing percutaneous coronary intervention for ST-elevation myocardial infarction (TRITON-TIMI 38): double-blind, randomized controlled trial Lancet 373, 723–31. 22. Anderluh, M., and Dolenc, M.S. (2002) Thrombin receptor antagonists; recent advances in PAR-1 antagonist development Curr Med Chem 9, 1229–50.

Chapter 9 Antithrombotic Effects of Naturally Derived Products on Coagulation and Platelet Function Shaker A. Mousa Abstract To date, there have been few systematic studies of the antiplatelet and/or anticoagulant effects of natural products. According to the Natural Medicines Comprehensive Database, approximately 180 dietary supplements have the potential to interact with warfarin, and more than 120 may interact with aspirin, clopidogrel, and dipyridamole. These include anise and dong quai (anticoagulant effects); omega 3-fatty acids in fish oil, ajoene in garlic, ginger, ginko, and vitamin E (antiplatelet properties); fucus (heparin-like activity); danshen (antithrombin III-like activity and anticoagulant bioavailability); and St. John’s Wort and American Ginseng (interference with drug metabolism). Other supplements, such as high doses of vitamin E (vitamin K antagonist activity), alfalfa (high-vitamin K content), and coenzyme Q10 (vitamin K-like activity), may affect blood clotting, which is dependent on vitamin K. Studies are needed to understand the role of various dietary supplements in thrombosis and their interactions with standard anticoagulants and antiplatelet drugs. Key words: Dietary supplements, natural products, anticoagulant, antiplatelet, antithrombotic, herbal–drug interaction, dietary supplement–drug interaction, thrombosis, hemostasis, bleeding, cardiovascular diseases, cerebrovascular diseases.

1. Introduction Cardiovascular and cerebrovascular diseases are the leading causes of death worldwide. The United States alone has an estimated 4 million patients on long-term antithrombotic therapies (anticoagulants with or without antiplatelet drugs), and this number is expected to increase. Surveys suggest that 15–25% of the general population uses dietary supplements. Recently, 43% of veterans administration (VA) ambulatory care patients reported using one or more dietary supplements, with up to 45% of the supplements S.A. Mousa (ed.), Anticoagulants, Antiplatelets, and Thrombolytics, Methods in Molecular Biology 663, DOI 10.1007/978-1-60761-803-4_9, © Springer Science+Business Media, LLC 2003, 2010

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taken having potential interactions with medications. Nonprescription drugs, vitamins, minerals, botanicals, homeopathies, and other complementary and alternative therapies are being actively promoted, resulting in an increased use of these products. Because antithrombotic therapies have narrow therapeutic windows, they must be monitored so that patients are not put at risk for thromboembolic or hemorrhagic complications. Drug interactions with herbal and other dietary supplements are much more difficult to capture, characterize, and predict because these products are not required to undergo FDA review or approval prior to marketing. They also do not need to meet the same quality standards as prescription drugs. Manufacturers are responsible for showing simply that they are safe and efficacious. Based on the pharmacodynamic and pharmacokinetic properties of commercially available supplements and herbal remedies, the potential for interactions is high. Although practitioners are encouraged to report such interactions to the FDA, published case reports of interactions are limited. Clinical guidance for practitioners who prescribe antithrombotic medications and patients who receive them with respect to the use of dietary supplements is lacking. Thus, there appears to be a need for increased awareness among practitioners about the potential harm or benefit of dietary supplements among patients receiving long-term antithrombotic therapies. Although a variety of dietary supplements may affect hemostasis, very few are absolutely contraindicated in people with bleeding disorders or on antithrombotic therapies. The medical community would benefit from evaluation and information on these supplements and their harm or benefit to antithrombotic therapies. Various substances have been proposed and approved as effective antithrombotics. These agents have been classified as inhibitors of platelet aggregation, the primary causes of arterial thrombosis, or coagulation, which causes primarily venous clots. Platelet aggregation inhibitors are designed to target various platelet activation mechanisms, including the thromboxane A2 and ADP receptor pathways, thrombin receptors, and glycoprotein (GP)IIb/IIIa receptors. Anticoagulant agents function primarily by inhibiting the production of fibrin and fibrin accumulation within venous blood. Anticoagulant therapies target thrombin, which converts fibrinogen to fibrin, either directly or indirectly. A significant amount of research has focused on the individual effects of antiplatelet and anticoagulant agents in treating and preventing thrombosis. Although studies have shown synergistic relationships between aspirin and heparin in reducing cardiovascular events (1, 2), little research has been conducted on the effects of combination therapies between platelet and coagulant

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inhibitors and their roles as antithrombotic agents. It has been shown that there is a synergistic relationship between aspirin and warfarin in decreasing the risk of embolism among patients with prosthetic heart valves, but the effects of combination therapy in direct relation to thrombosis were not clarified (3). In addition, the effects of combining naturally occurring antiplatelet and anticoagulant agents have yet to be determined. The relationship between cancer and the activation of platelets and coagulation has been established. Tumor cell-induced platelet aggregation not only plays an important role in thrombosis among cancer patients, but is also recognized as a significant step in the metastatic cascade (4). Likewise, tumors have been shown to activate coagulation by increasing plasma fibrinogen and fibrin monomer (5).

2. Flavonoids 2.1. Resveratrol

Resveratrol (3,4’,5-trihydroxystilbene), a naturally occurring flavonoid found primarily in grapes, red wine, and peanuts (6, 7), has been shown to have antiplatelet properties. It is thought that resveratrol inhibits platelet aggregation induced by collagen, thrombin, and ADP (8). The effects of resveratrol on platelet aggregation seem to be dependent on concentration, and inhibitory results have been observed in vitro and in vivo (8). Despite evidence of its ability to inhibit thrombin-induced platelet activity, resveratrol has not been shown to affect thrombinassociated fibrin production or posses any other anticoagulant properties. Furthermore, resveratrol has yet to be linked to the inhibition of platelet aggregation induced by tumor cells. It has, however, shown promise as an anticancer agent, independent of its ability to inhibit activated platelets. A recent study showed that flavonoids were able to trigger apoptosis in human leukemia and breast cancer cells (9). The idea of using resveratrol as a possible line of treatment in cancer is still in the developmental stage, and more research is needed (9).

2.2. Tea Extract

A major component in green tea, epigallocatechin-3 gallate (EGCG) is believed to be the active ingredient involved in platelet inhibition in humans (10). EGCG primarily targets thrombininduced platelet activation, and has been shown to decrease the concentration of the calcium ionophore A23187 (10, 11). Platelet aggregation induced by collagen, thrombin, ADP, and epinephrine is also inhibited by EGCG (11, 12). Catechins present in green tea have been found to significantly inhibit the binding of fibrinogen to the platelet surface GPIIb/IIIa complex in humans by decreasing the levels of cytoplasmic calcium

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(11), and it is assumed that EGCG plays a major role in this inhibitory activity. Green tea stands as a promising antiplatelet agent, more so than an anticoagulant, as it does not affect coagulation activities, including prothrombin and thrombin time, in human citrated plasma (11, 12). Although green tea does not play a role in preventing coagulation, it does act as an anticancer agent (13). The anticancer activity of green tea catechins, including EGCG, that possess the gallate group could be mediated in part through their ability to suppress the tyrosine kinase activity of the platelet-derived growth factor (PDGF)-β receptor (14). The effectiveness of green tea in inhibiting platelet aggregation might also be dependent on the form of the tea. It has been suggested that commercially processed tea, in contrast to unprocessed, dried tea leaves, does not decrease thromboxane levels in rats (15). Furthermore, only green tea seems to have an antithrombotic effect, while black tea fails to evoke similar effects (16). 2.3. Genistein

Found predominantly in soy products, genistein acts as a tyrosine kinase inhibitor and, according to some indications, an inhibitor of platelet aggregation (17, 18). The primary mechanism of inhibition of platelet function by genistein is the reduction of cytosolic free calcium concentration (18, 19). Genistein has also been shown to inhibit collagen- and thromboxane analog U46619induced platelet aggregation (17, 20). In addition to its ability to inhibit activated platelets, genistein has been shown to induce cell cycle arrest and apoptosis, as well as inhibit the growth of cancer cells derived from specific cancers of the head, neck, breast, lung, and prostate in culture (21). Genistein also appears to be an effective anti-angiogenic agent with respect to tumor growth (22), but there is no evidence that the anticancer activity of this flavonoid includes inhibition of cancer-induced platelet activation. There is conflicting data on the ability of genistein to inhibit thrombininduced release of serotonin secretions (18, 20). Thus, while it is clear that genistein has potential antiplatelet uses, evidence of any anticoagulant activity is lacking.

2.4. Echistatin

A protein derived from snake venom, echistatin is a 5,000-Da disintegrin capable of platelet inhibition (23–25). This disintegrin acts as a competitive inhibitor of platelet αIIbβ3 integrin binding to fibrinogen (23, 26), and decreases phosphorylation to attenuate platelet adhesion (25). There is also evidence that echistatin plays a role in the inhibition of collagen (27). To date, while the anticoagulant potential of echistatin has yet to be demonstrated, the ability of echistatin to irreversibly bind to integrin αIIbβ3 implicates this disintegrin in the treatment of tumorinduced angiogenesis and tumor cell metastasis (24). Echistatin may also play a pivotal role in the disassembly of focal adhesions in fibronectin-adherent B16-BL6 melanoma cells by reducing the

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levels of pp125FAK tyrosine phosphorylation (28). Currently, a solid link between the anticancer activity of echistatin and the inhibition of cancer-induced thrombosis has yet to be demonstrated. 2.5. Fisetin

Fisetin is a naturally occurring purified protein kinase C inhibitor that can inhibit platelet aggregation (29). Platelet activity is primarily suppressed through complete blockage of thrombininduced shape changes (29, 30). It has been suggested that the mechanism of action of fisetin involves inhibition of thromboxane formation and thromboxane receptor antagonism (30). Fisetin inhibits cathepsin G-induced platelet aggregation as well (29). Fisetin has been shown to inhibit the proliferation of normal and cancer cells and induce apoptosis of human promyeloleukemia cells (31, 32). The anticancer properties of fisetin do not appear to extend to cancer-induced platelet aggregation or coagulation. Although current findings support the use of fisetin as an antiplatelet agent, research to date has not focused on the potential anticoagulant properties of this flavonoid.

3. Garlic 3.1. Allicin

The sulfuric compound responsible for the distinct odor of garlic, allicin, appears to be primarily responsible for the inhibitory effect of garlic on platelet aggregation. Derived from the cleavage of alliin by alliin lyase (33), allicin inhibits platelet activity in vitro without affecting cyclooxygenase, lipoxygenase, thromboxane, vascular prostacyclin synthase, or cyclic AMP levels (34, 35). The exact mechanism by which allicin inhibits platelet aggregation is unclear, but may be similar to that of ajoene. Ajoene is a rearrangement of allicin that inhibits platelet aggregation in vitro through inhibition of granule release and fibrinogen binding (36). Ajoene is capable of irreversibly inhibiting platelet aggregation induced by arachidonic acid, adrenaline, collagen, ADP, and the calcium ionophore A23187 (37). In addition to their antiplatelet capabilities, allicin and ajoene have been shown to exhibit antitumor properties. Allicin, but not alliin, inhibits the proliferation of human mammary (MCF-2), endometrial, and colon (HT-29) cancer cells (38). In addition to inhibiting proliferation, ajoene also induces apoptosis in human CD34-negative leukemia cells (39). Currently, allicin shows potential antiplatelet activity.

3.2. Diallyl Trisulfide

Diallyl trisulfide is an important paraffinic polysulfide component of garlic that exhibits certain reversible antiplatelet functions (40).

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Studies have clearly indicated that this polysufide inhibits platelet aggregation and calcium ion mobilization induced by thrombin in a concentration-dependent manner (41). Diallyl trisulfide also appears to inhibit thromboxane A2 synthesis, except in a cell-free system (34, 40, 41). No effects of diallyl trisulfide on coagulation have been documented; however, evidence of chemopreventative effects on tumorogenesis have been reported (42). It is thought that diallyl trisulfide inhibits cancer progression by augmenting the activation of T cells and enhancing the antitumor function of macrophages (43). However, these properties do not seem to effect or prevent cancer-induced platelet aggregation or coagulation. 3.3. Allyl Disulfide

There is very little research on the effects of allyl disulfide on platelet aggregation. It has been suggested that this polysulfide inhibits thromboxane A2 , similar to other sulfides present in garlic (34). However, the lack of data on allyl disulfide suggests that this component plays a minor, if any, role in the antiplatelet activities of garlic.

4. Antioxidants 4.1. Vitamin E

Along with its antioxidant properties, vitamin E has multiple antiplatelet properties that appear to be independent of its antioxidant activities (44, 45). Vitamin E has been shown to decrease platelet adhesion to collagen, fibrinogen, and fibronectin, and increase platelet sensitivity to prostaglandin E1 (46–49). Vitamin E also induces the inhibition of protein kinase C (44, 45). Evidence suggests that vitamin E affects phospholipase A2 and at least one other step in the thromboxane A2 cascade, but that inhibition of platelet aggregation is not exclusively dependent on these steps within the cascade (50, 51). The reversible effects of vitamin E on platelet activity are concentration dependent up to maximal uptake by the platelet, at which point excess vitamin E has no effect on function (46, 52). A synergistic effect of vitamin E and other antiplatelet therapies has been suggested to cause an increase in bleeding (45). Among the numerous mechanisms of inhibition of platelet activity by vitamin E, there is no evidence to suggest that this vitamin has anticoagulant properties. Vitamin E may function as an anticancer agent, as it has been shown to inhibit cell proliferation and monocyte adhesion (53). In fact, an early study proposed that supplemental antioxidants such as vitamin E could impede tumor dissemination caused by platelet aggregation (54). Although it has been suggested that the anticancer properties of vitamin E are due to its ability to inhibit platelet aggregation, solid evidence of the reverse does not exist.

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4.2. Selenium

A deficiency in the trace element selenium enhances platelet activating factor and increases the risk of atherosclerosis (55, 56). Therapies aimed at increasing the levels of selenium in the body result in the inhibition of platelet aggregation, primarily through thromboxane A2 (57–59). Increases in selenium result in the inhibition of thromboxane A2 synthesis in a concentration-dependent manner in vitro and in vivo and a concomitant decrease in the amount of thromboxane B2 released (52, 58, 59). Increased levels of selenium do not affect the biosynthesis of prostaglandin or platelet adherence to fibrinogen (48, 57). Some studies have suggested that selenium may reduce the risk of cancer when properly incorporated into the diet (60) and that it may have chemopreventative effects in breast cancer (61). The specific mechanisms underlying the anticancer properties of selenium are as yet undefined, as are the anticancer effects on platelet activation and coagulation. In addition, there is no evidence to date that selenium functions as an anticoagulant.

4.3. Methylsulfonylmethane (MSM)

The dietary supplement methylsulfonylmethane (MSM) has multiple potential uses, including treatment of athletic injuries, bladder disorders, and pain syndrome (62), but studies tend to focus disproportionately on its affects on arthritis and seasonal allergic rhinitis (SAR) (63, 64). MSM is effective in reducing the symptoms of SAR with relatively low toxicity (2,600 mg/day for 30 days) and few side effects, which promotes the safety of MSM (63). MSM also seems to function similarly to aspirin as a cancer chemopreventive drug, inducing the differentiation of murine erythroleukemia cells (64). Under differentiation-inducing conditions and at concentrations reported in other studies, MSM did not affect prostaglandin E2 or cyclooxygenase activity (64). Thus, if MSM does posses antiplatelet properties, it most likely affects aspects of platelet aggregation other than prostaglandin or cyclooxygenase. There is no current evidence of antiplatelet or anticoagulant properties of MSM nor has the role of MSM in human nutrition been thoroughly studied (62).

5. Anticoagulants 5.1. Heparin

Heparin is a naturally occurring indirect thrombin inhibitor in the body and has been utilized as an anticoagulant for years. The mechanism of action of heparin consists of increasing the activity of antithrombin III to inhibit thrombin synthesis and ultimately the production of fibrin (65). However, some studies suggest that in addition to acting on antithrombin III, heparin may

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directly affect thrombin (66). Heparin has been proven to prevent arterial thrombosis; however, the necessary dosage to do so greatly increases activated partial thromboplastin time (aPTT) (67). Although its ability to prevent arterial thrombosis suggests that heparin possesses antiplatelet properties, studies indicate that heparin is more efficacious when used in the treatment of venous thrombosis (67). Heparin is also indicated as a potential cancer therapeutic. One study in particular suggested that heparin, combined with IL-2/LAK therapy, may be useful in preventing fibrin coagulation on tumor cells (68). This study also suggested a possible relationship between the reduction of fibrin coagulation initiated by tumor cells and the anticoagulant activity of heparin. The anticoagulant response produced by heparin is often unpredictable, and a major inducer of thrombus growth, namely fibrin-bound thrombin, is unaffected by heparin (69). A major drawback to heparin use is the development of heparin-induced thrombocytopenia, an immunoglobulin-mediated reaction associated with an increased risk of thrombotic complications (70). Direct thrombin inhibitors, however, are capable of inhibiting fibrin-bound thrombin and provide an alternative treatment for patients who have developed heparin-induced thrombocytopenia (69). The undesired effects and limitations of heparin use have led to the development of several alternative heparin derivatives. 5.2. Hirudin

One of the most important naturally occurring thrombin inhibitors is hirudin, a salivary extract of Hirudo medicinalis (71). Hirudin acts directly on thrombin by binding to several sites, inhibiting all of its functions, including fibrin-bound thrombin (69, 72). Unlike heparin, hirudin is associated with a predicable response and does not cause further thrombotic complications, such as thrombocytopenia (73). Hirudin does not appear to cross-react with heparin or heparin derivatives (72). The only concern with hirudin use is a high risk of bleeding (74). Derivatives of hirudin are currently in development and show promise as potent inhibitors of fibrin deposition on clot surfaces, platelet deposition, and thrombus formation (75). Polyethylene glycol– hirudin may also prove to be an effective agent in treating arterial thrombosis (76). The primary use of hirudin currently is in venous thrombosis; it has yet to be evaluated for the treatment of arterial thrombosis. Hirudin diminishes the metastatic potential of tumor cells in the presence and absence of fibrinogen, suggesting the potential of direct thrombin inhibitors to serve as anticancer agents. Hirudin was able to prevent platelet aggregation induced by human chlorangiocarcinoma (CCA) cells via thrombin, suggesting that it may prove useful in preventing tumor cell-induced platelet aggregation or metastasis in CCA (4).

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The impact of dietary supplements on normal hemostasis and antithrombotic therapy should be given careful consideration. Supplements that have been reported to affect normal coagulation and platelet activity and/or have been reported to possibly interact with coumarin anticoagulants include danshen (Salvia miltiorrhiza Bunge, Lamiaceae), garlic (Allium sativum L., Lilliaceae), ginkgo (Ginkgo biloba L., Ginkgoaceae), American ginseng (Panax quinquefolius L., Araliaceae), Asian ginseng (Panax ginseng C.A. Meyer), and St. John’s wort (Hypericum perforatum L., Clusiaceae). However, most of these reports are either theoretical or consist of individual cases. In addition to supplement heterogeneity, individual patient responses reflecting specific genetic polymorphisms in the cytochrome P450 enzyme system and other metabolic pathways can alter the metabolism of warfarin (a synthetic dicoumarol, often sold under the trade R name Coumadin ) and/or dietary supplements. Given the variety of dietary supplements available, an important aspect of the management of patients on oral anticoagulant therapy is regular assessment of supplement use to ensure that patients are aware of the potential risks and benefits of taking supplements in conjunction with their prescribed medications.

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Chapter 10 Assessment of Anti-Metastatic Effects of Anticoagulant and Antiplatelet Agents Using Animal Models of Experimental Lung Metastasis Ali Amirkhosravi, Shaker A. Mousa, Mildred Amaya, Todd Meyer, Monica Davila, Theresa Robson, and John L. Francis Abstract It is well established that the blood coagulation system is activated in cancer. In addition, there is considerable evidence to suggest that clotting activation plays an important role in the biology of malignant tumors, including the process of blood-borne metastasis. For many years our laboratory has used experimental models of lung metastasis to study the events that follow the introduction of procoagulantbearing tumor cells into circulating blood. This chapter focuses on the basic methods involved in assessing the anti-metastatic effects of anticoagulants and anti-platelet agents using rodent models of experimental metastasis. In addition, it summarizes our experience with these models, which collectively suggests that intravascular coagulation and platelet activation are a necessary prelude to lung tumor formation and that interruption of coagulation pathways or platelet aggregation may be an effective anti-metastatic strategy. Key words: Anticoagulants, metastasis, experimental models, tissue factor, anti-platelet drugs.

1. Introduction 1.1. Coagulation Activation in Cancer

Malignancy is associated with activation of the coagulation system (1) and patients with cancer are at significantly increased risk of developing venous thrombosis (2). A link between cancer and thrombosis was first recognized by Bouillaud in 1823 (3). In 1865, Armand Trousseau observed a high incidence of thromboembolic disease (TED) in patients with gastric carcinoma and further suggested that thrombophlebitis may be symptomatic of cancer of other internal organs (4). Substantial clinical, laboratory, pharmacological, and histological evidence has since

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been accumulated in support of this relationship. Currently, the term Trousseau’s syndrome is used to describe any type of coagulopathy associated with malignancy ranging from venous and/or arterial thromboembolism to chronic disseminated intravascular coagulation (DIC) with or without thrombotic microangiopathy or secondary hyperfibrinogenolysis (5, 6). It is now well established that many cancer patients exhibit a hypercoagulable state or low-grade DIC. In general, clotting changes are rarely marked, usually asymptomatic, and more commonly associated with diffuse thrombosis rather than bleeding. The most common hemostatic abnormalities are hyperfibrinogenemia and thrombocytosis which are found in 50–80% and 10–57% of cancer patients, respectively (7–10). Thrombocytopenia is less common and usually occurs due to cytotoxic therapy or bone marrow involvement. Routine coagulation screening tests such as prothrombin time (PT) and activated partial thromboplastin time (APTT) are not usually helpful in demonstrating a hypercoagulable state in cancer patients. Unequivocal detection of hypercoagulability can be achieved with sensitive molecular markers of coagulation activation such as fibrinopeptide A (FpA) or thrombin–antithrombin (TAT) complex. Elevated levels of these markers are observed in up to 90% of cancer patients (11). Interestingly, FpA levels increase as the patients become terminally ill and persistently elevated levels may indicate treatment failure and a poor prognosis (12). Finally, many patients have an increased rate of coagulation that can be detected with relatively simple whole blood coagulation tests such R as Thromboelastography (TEG) and SonoclotTM analysis (13). 1.2. The Role of Tissue Factor in Cancer-Associated Coagulation Activation

Although the mechanisms by which coagulation is activated during malignancy are multifactorial, tissue factor (TF), the primary initiator of coagulation, has been recognized to play an important role in this process. Aberrant expression of TF has been associated with various pathological conditions, including cancer. Many tumor cell types constitutively express TF on their surfaces and may trigger the production of TF by adjacent host cells (monocytes and endothelial cells) (14). Physiologically, TF expression is limited to extravascular sites such as subendothelial layers of the vessel wall. However, two forms of circulating TF have been described: an alternatively spliced soluble protein (15), and a form associated with cell-derived microparticles (MP) (16). The contribution of the latter type of TF to a prothrombotic state in cancer has been suggested. High levels of circulating TF have been found in the plasma of patients with different cancer types and correlated with other coagulation activation markers (17, 18). A recent case study showed extremely high levels of MP-associated TF in the plasma of a lung cancer patient with a severe form of Trousseau’s syndrome (19). Although TF

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activity in the patient’s plasma did not correlate with the high levels of circulating TF antigen, a recent study from the authors’ laboratory showed that TF-dependent procoagulant activity was correlated with TAT levels in cell-free plasmas of mice with growing orthotopic pancreatic human tumors, supporting the hypothesis that circulating tumor-derived TF causes coagulation activation in vivo (20). 1.3. The Role of Tissue Factor and Thrombin in Tumor Growth, Angiogenesis, and Metastasis

The effects of cancer on the coagulation protease cascade do not constitute a unidirectional relationship. Rather, a large body of evidence suggests that components of the hemostatic system promote tumor growth, angiogenesis, and metastasis, suggesting that activation of the clotting pathways in cancer patients is not merely an epiphenomenon of the disease. One potential pathway involves TF-mediated clotting activation, which leads to thrombin generation (locally or systemically), platelet activation, and tumor-associated fibrin formation. TF also appears to regulate, directly or indirectly, tumor angiogenesis via mechanisms independent of clotting activation. In addition, the extracellular functions of the TF/VIIa complex appear to cooperate with the signaling functions of the TF cytoplasmic domain to support blood-borne metastasis (21, 22). The information currently available on the multiple effects of the TF pathway on tumor pathophysiology provides the basis for considering TF as a target for anti-metastatic and anti-angiogenic therapy. Thrombin is a multi-functional serine protease that is rapidly generated following clotting activation. In addition to its direct role in fibrin formation and platelet activation, which enhance tumor metastasis, thrombin can promote angiogenesis via direct as well as indirect mechanisms. For example, thrombin can potentiate vascular endothelial growth factor (VEGF) activity on endothelial cells by up-regulating the expression of VEGF receptors on endothelial cells (23). Both thrombin and VEGF can in turn stimulate DNA synthesis in endothelial cells, either alone, or in a synergistic manner (24).

1.4. The Role of the Coagulation System in Blood-Borne Metastasis

For many years, research in the authors’ laboratory has focused on the events that take place when cancer cells enter the circulating blood (11). Specifically, we have investigated whether coagulation activation induced directly by tumor cells is important in hematogenous (blood-borne) metastasis. To address this issue, we have utilized experimental models of metastasis. In this type of model, TF-expressing tumor cells are injected intravenously into the tail vein of mice or rats. Injected tumor cells immediately become entrapped in the microvasculature of the lung – “the organ of first encounter.” Consequently, evidence of intravascular coagulation can be detected shortly after tumor cell injection. This includes a fall in platelet count and fibrinogen and Factor

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X (FX) levels and the presence of increased plasma hemoglobin levels. The latter heralds the onset of microangiopathic hemolytic anemia caused by formation of fibrin strands in the microvasculature. Animals injected with tumor cells subsequently develop secondary tumors in the lungs. It is important to note that animal models of experimental metastasis do not completely represent human metastasis, since they do not include stages of the metastatic processes that occur prior to tumor cell entrance into the blood stream (e.g., migration, invasion, and intravasation). However, these models serve to isolate the events of interest in the circulating blood and thus are useful for proof of concept studies. In this current chapter, we aim to: (a) describe a number of methods that are useful in assessing the effects of anticoagulants or anti-platelet agents on tumor cell-induced clotting activation and experimental lung metastasis; (b) describe the results obtained from various such agents and provide some perspective on how such data could be interpreted.

2. Materials and Methods 2.1. Tumor Cells

Human or murine tumor cells are cultured in their appropriate media until 70% confluent. Cells are then harvested, preferably with a non-enzymatic cell dissociation solution (Sigma), washed, and suspended in phosphate-buffered saline (PBS) at the desired concentrations. It is important for the final cell suspension to be free of visible aggregates.

2.2. Intravenous Injections

The recommended intravenous injection volume of cell suspensions or reagents is 0.1–0.2 ml for mice, although up to 0.5 ml could be injected slowly (∼ 30 s). Before injections, animals are warmed for 3–5 min using a heat lamp. They are then placed in a standard restrainer and are injected in the lateral tail vein (Fig. 10.1a). In our studies, the number of tumor cells injected in one animal ranged from 1×105 to 2×106 cells. Typically, in order to observe marked tumor cell-induced clotting activation, 1–2×106 cells were injected. However, for experimental metastasis studies, 1×105 – to 1×106 cells were given.

2.3. Blood Draws

Blood samples are obtained by cardiac puncture of anesthetized animals (Fig. 10.1b). Exactly 0.5 ml of blood is collected into a 1-ml syringe containing 0.1 ml of 3.2% trisodium citrate using a 27- or 25-gauge needle for mice or rats, respectively.

2.4. Evidence of Clotting Activation

In our studies we used platelet count, FX and fibrinogen concentrations, and plasma hemoglobin levels as indicators of tumor

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Fig. 10.1. (a) Injection of tumor cells into the tail vein of experimental mice. Cells injected in this manner are arrested rapidly in the microvasculature of the lungs – the organ of first encounter – and activate blood coagulation. (b) Blood (0.5 ml) is collected by cardiac puncture into a syringe containing trisodium citrate.

cell-induced clotting activation. Platelet counts were measured automatically using an electronic Coulter Counter no later than 10 min after blood draws. The blood was then centrifuged at 5,000 rpm for 10 min. The cell-free plasma was separated and frozen at –70◦ C until analysis. FX and fibrinogen levels were measured by standard automated coagulation techniques. Plasma hemoglobin levels were measured by the orthotolidine colorimetric assay (25) modified for use in microtitre plates. A range of hemoglobin concentrations between 5 and 100 μg/ml was prepared by dilution of a standard hemoglobin solution (BDH Chemicals). Animal plasmas were diluted 1:5 in PBS. To each well of a microtitre plate were added 130 μl of orthotolidine solution (0.25% [w/v] orthotolidine in 90% [v/v] glacial acetic acid) and 5 μl of diluted standard solution or test plasma. The reagents were mixed and after 2 min, 130 μl of 1.2% hydrogen peroxide were added to each well. After 10 min at room temperature, 40 μl of glacial acetic acid were finally added and absorbances were immediately measured at 630 nm using a plate reader. 2.5. Quantitation of Pulmonary Tumor Nodules

Fourteen to forty-two days (depending on the cell line used) after tumor cell injection, animals were euthanized and the lungs were dissected en bloc from the thoracic cage, rinsed in water, and placed in Bouin’s fixative solution (Sigma). On occasions when there are many surface nodules present, enumeration of tumor foci can be made easier if the lungs are insufflated with the fixative. Briefly prior to dissection of the lungs, 2–3 ml of Bouin’s solution is injected via the trachea, thus insufflating the lungs. The lungs are then dissected out as above, rinsed to remove excess fixative, and evaluated macroscopically.

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3. Summary of the Results Obtained from Models of Experimental Metastasis 3.1. Effects of Heparins and Coumadin

We first set out to assess the effects of the commonly used anticoagulants heparin and coumadin in a rat model of experimental metastasis (26–29). Unfractionated heparin was administered intravenously 1 h before intravenous injection of MC28 fibrosarcoma cells. Coumadin was given in the animals’ drinking water, starting 1 week before tumor cells were injected to allow anticoagulation to achieve therapeutic levels (PT at least twice that at baseline). In control (no anticoagulant therapy) animals, evidence of tumor cell-induced coagulation was observed within 30 min of tumor cell injection. These changes were almost completely suppressed in anticoagulated animals. In addition, systemic anticoagulation significantly reduced lung tumor formation compared with controls. Delaying warfarinization almost abolished the antimetastatic effect of coumadin, suggesting that the important effects in this model occur at an early stage post-tumor injection (30). We then investigated whether the anti-metastatic effect of anticoagulants was due to inhibition of tumor cell growth in the lungs or whether anticoagulation reduced the physical trapping of tumor cells in the pulmonary microvasculature. To address this, we conducted fate studies of radiolabeled tumor cells in control and anticoagulated animals (29). The results showed that the antimetastatic effects of heparin and coumadin in this model were not due to inhibition of initial tumor cell trapping in the lung, but were due to increased subsequent clearance of the cells from the pulmonary microvasculature. These data indicated that, in the presence of competent coagulation pathways, tumor cells trigger thrombin generation and form complexes with platelets and fibrin that potentially aid adherence to the vascular endothelium, an event that is a pre-requisite to the important metastatic step of extravasation. Anticoagulation reduced the ability of tumor cells to activate coagulation and platelets and hence to form sticky aggregates. Thus they were cleared more rapidly from the lung, before they had the opportunity to form tumors. These events are summarized in Table 10.1. Our results are consistent with findings of Palumbo and colleagues who demonstrated a reduced retention of radiolabeled tumor cells injected into fibrinogendeficient mice (31). Interestingly, these authors further demonstrated that inhibition of thrombin by hirudin was found to

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Table 10. 1 Effect of anticoagulation on the intravascular events following tail vein injection of tumor cells in experimental animals Normal coagulation

Anticoagulated

Cell trapping

Cell trapping

Platelet aggregation

No platelet aggregation

Fibrin formation

No fibrin formation

Delayed clearance

Rapid clearance

Extravasation

Cell death

Tumor formation

No tumor formation

further reduce the already low metastatic potential of tumor cells in fibrinogen-deficient mice. It was thus concluded that fibrin(ogen) is an important determinant of metastatic potential and that thrombin contributes to the metastatic process through at least one fibrinogen-independent mechanism. In our study fibrin-platelet microthrombi were observed by electron and light microscopy 30–60 min after intravenous injection of tumor cells. Fibrin was observed adjacent to tumor cells, but it was not clear from these experiments whether fibrin generation occurred on the surface of fibrosarcoma cells. Rapid accumulation of radiolabeled platelets at the site of tumor lodgement supported the microscopic observations. 3.2. Anti-metastatic Effect of a Non-anticoagulant Low-Molecular Heparin (LMWH) Versus the Standard LMWH Enoxaparin

Having observed the anti-metastatic effect of unfractionated heparin, we then tested the effects of the LMWH enoxaparin as well as a non-anticoagulant low molecular weight heparin (NA-LMWH) on tumor cell-induced clotting activation in vivo and experimental metastasis (32). NA-LMWH was prepared by fragmenting porcine mucosal heparin into LMWH followed by reduction with sodium borohydride and acid hydrolysis. NALMWH does not exhibit any inhibitory activity against factors Xa and IIa. However, it has the ability to cause the release of tissue factor pathway inhibitor (TFPI) from vascular endothelial cells in vitro similar to enoxaparin (Fig. 10.2). We therefore hypothesized that NA-LMWH will exhibit only a moderate anti-metastatic effect (compared to enoxaparin) without affecting tumor cell-induced clotting activation in vivo. In this study we used the B16 melanoma mouse model of experimental metastasis. The anticoagulant effect of enoxaparin and NA-LMWH (both at 10 mg/kg) was measured 3 h after subcutaneous or 15 min after intravenous (tail vein) injection of either drug

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NA-LMWH Enoxaparin 200

100

0 0.01

0.05

0.10 0.50 1.00 3.00 Concentration (μg/106 cells)

10.00

Fig. 10.2. Both enoxaparin and a non-anticoagulant low molecular weight heparin (NA-LMWH) have the ability to cause the release of TFPI from endothelial cells in vitro.

in experimental mice. Blood samples were collected by cardiac puncture into trisodium citrate and whole blood coagulation was assessed by TEG and by a Sonoclot Analyzer (Sienco, USA). (APTT); anti-Xa, and anti-IIa activity of the heparins in mouse platelet-poor plasmas were also measured using an automated coagulation analyzer. For metastasis experiments, mice were injected subcutaneously with a bolus of saline or enoxaparin or NA-LMWH and 4 h thereafter all mice were injected intravenously with B16 tumor cells. The mice then received daily doses (for 14 days) of either heparin. Experimental lung metastasis was assessed 15 days after tumor cell injection. As expected, enoxaparin, but not NA-LMWH, exhibited significant anti-Xa and anti-IIa activities. Intravenous injection of melanoma cells resulted in a significant and rapid reduction (>50%) in the platelet count of control mice previously injected with saline. This fall in platelet count was abolished in mice treated with enoxaparin prior to tumor cell injection. In contrast NA-LMWH had no effect on tumor cell-induced thrombocytopenia (Fig. 10.3). Both enoxaparin and NA-LMWH reduced experimental metastasis by 70% (P<0.01, Fig. 10.4). In the context of other interventional studies involving the anticoagulants mentioned above, the significant anti-metastatic effect of NA-LMWH is intriguing and points to anti-metastatic mechanisms that are independent of coagulation and platelet activation pathways. The anionic properties of heparin and its fractioned derivatives are thought to be responsible for heparins’ anti-cancer effects, including angiogenesis, tumor cell adhesion,

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Platelet numbers (x106/ml)

1200 1000 800 P < 0.01

600

P < 0.01

400 200 0 Baseline

Control

Enoxaparin

NA-LMWH

Fig. 10.3. The effect of enoxaparin versus NA-LMWH on tumor cell-induced thrombocytopenia. Unlike enoxaparin, due to its lack of anticoagulant activity, NA-LMWH did not reverse the fall in platelet count caused by the injection of tumor cells.

# of lung tumor nodules

PBS 200

150

ENOXAPARIN P < 0.01

100

P < 0.01

NA-LMWH

50

0

PBS

Enoxaparin

NA-LMWH

Fig. 10.4. Right panel: The effect of enoxaparin versus NA-LMWH on experimental lung metastasis. Left panel: Representative lungs of control and heparin-treated mice 2 weeks after the injection of B16 tumor cells.

and malignant transformation. Possible coagulation-independent mechanisms for inhibition include binding of heparin to angiogenic growth factors (such as basic fibroblast growth factor and VEGF) and modulation of TF (33–35). A key component of metastasis is the adhesion of circulating tumor cells to the vascular endothelium of organs distant from the primary tumor site. P-selectin-mediated tumor cell interactions have been shown to promote metastasis (36, 37). Expression of carbohydrate moieties required for P-selectin binding is associated with increased metastasis and poor survival in various tumor cell types (38–40).

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Furthermore, it has been demonstrated that heparins (unfractionated and low molecular weight) and derivatives (fondaparinux) exert differential anti-metastatic effects at comparable anticoagulant activities in vivo as well as differential inhibitory effects on P-selectin-mediated interactions (37). It may therefore be possible to suggest that the degree of inhibition of P-selectin function of a given anticoagulant may provide a parameter to indicate its anti-metastatic potential. In order to examine the role of tumor cell TF on experimental metastasis, we used the monoclonal antibody H36 (Sunol Molecular Inc, Miramar, FL), which competitively inhibits human TF/VIIa-dependent FX activation (11). In this study we injected intravenously the TF-expressing C15 (metastatic) variant of A375 human melanoma cells in athymic nude mice (nu/nu). The antibody was diluted to concentrations of 0.1–3 mg/ml, and 0.2 ml of each concentration were injected intravenously 10 min before injection of tumor cells and at several time points (3, 7, 10, 14, 17, 21, 28, and 35 days) thereafter. The experiment was terminated 42 days after tumor cell injection. All doses of H36 antibody were well tolerated by the experimental animals. No bleeding or any other adverse effects were observed as a result of the antibody injection. H36 inhibited experimental lung metastasis at all doses tested (Fig. 10.5). There was a relationship between the antibody dose and lung tumor formation insofar as all eight animals given 0.1 mg/kg (the lowest dose) had metastasis and this group had the highest total number

H36

Control 140 Number of Lung Tumors

3.3. The Effect of TF Inhibition on Experimental Metastasis

120 100 80 60 40 20 0 Control

0.1

1

3

10

30

H36 (mg/kg)

Fig. 10.5. Effects of the anti-human TF monoclonal antibody H36 on experimental lung metastasis. Representative lungs taken from control and antibody-treated mice are included. Tumors appear as pale nodules.

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of lung nodules. However, only five of nine animals receiving the highest dose (30 mg/kg) had observable surface metastatic foci and, as a group had the lowest number of nodules. In a previous study, we preincubated the same tumor cells with or without two anti-TF antibodies, H36 and I43 (which blocks the binding of VII(a) to TF), before injecting the cells into experimental animals. In these experiments, we also demonstrated that tumor cell TF activity blockade is associated with a significant reduction in experimental metastasis. It is important to note that in our studies, only TF expressed by human tumor cells was inhibited by the anti-TF antibody, since H36 does not inhibit the function of murine TF (data not shown). This explains the absence of any bleeding side effects in our studies despite high doses of antibody administered. In a human trial, the H36 antibody caused spontaneous minor bleeding in a dose-related manner although the majority of those bleeding episodes were clinically consistent with platelet-mediated bleeding (41). Recently, Snyder and colleagues have developed an elegant mouse model in which expression of human TF is under the control of the murine TF promoter (42). These knock-in mice, referred to as TFKI, have human TF expression similar to murine TF in wild type animals and have normal hemostasis. Interestingly, when TFKI mice were treated with neutralizing anti-human TF antibody, cardiac bleeding was observed. However, the TFKI mouse may prove to be useful in assessing anti-tumor effects of anti-TF antibodies provided that tumors expressing human TF are introduced in to the host animal. Next we examined the anti-metastatic effect of the natural inhibitor of TF, TFPI, a plasma protein that regulates TFdependent reactions by neutralizing the catalytic activity of FXa and/or forming a quaternary inhibitory complex with TF, FVIIa, and FXa on the cell membrane (43, 44). TFPI’s anticoagulant function appears to be as efficient as unfractionated heparin (45). In our laboratory, we intravenously injected murine recombinant TFPI shortly before injection of B16 tumor cells in mice (46). This almost completely inhibited tumor cell-induced thrombocytopenia (Fig. 10.6a) and significantly reduced experimental metastasis (Fig. 10.6b). We then tested the effect of cellular TFPI expression on the metastatic potential of tumor cells. In order to achieve this, we transfected B16 cells (which do not express TFPI) with murine TFPI. This significantly reduced tumor cells TF-mediated procoagulant activity without altering TF antigen expression. When injected intravenously into mice, the transfected cells failed to produce a significant fall in platelet counts compared to control (non-transfected) or antisense transfected B16 cells. The TFPItransfected cells also formed significantly fewer lung tumor nodules compared to the above-mentioned control cells (Fig. 10.7).

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Fig. 10.6. (a) Intravenous injection of recombinant murine TFPI (rmTFPI) significantly inhibited tumor cell-induced thrombocytopenia caused post-injection of B16 melanoma cells. (b) Experimental lung metastasis was significantly inhibited by rmTFPI.

These results suggested that both circulating and tumor cellassociated TFPI appear to play a role in blood-borne metastasis. Consistent with the above findings, Hembrough et al showed a significant reduction in B16 experimental lung metastasis by

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Fig. 10.7. The effect of TFPI (sense, antisense, vector) transfection of B16 melanoma cells on cellular procoagulant activity as measured by a clotting assay. Fourteen sense (S), two antisense (AS) and one vector only cell lines were prepared and compared to untransfected wild-type B16 cells. S10 and AS1 were used for the metastasis experiments. Sense cell lines exhibited much lower procoagulant activity and caused the formation of significantly fewer lung tumor nodules (inset).

intraperitoneal injection of human TFPI (47). It is noteworthy that in the latter study TFPI was given daily after tumor cell injection. The authors also showed that another inhibitor of TF/VIIa, the nematode anticoagulant protein rNAPc2, had a similar antimetastatic effect compared with TFPI, whereas rNAP5, another nematode anticoagulant protein that specifically inhibits FXa did not exhibit significant antimetastatic effects. These results suggest that the proteolytic activity of TF/VIIa may promote experimental metastasis by mechanisms independent of FXa activation. 3.4. Effect of Anti-platelet Agents on Experimental Metastasis

Evidence suggests that platelets are an integral part of the microthrombus that enhances the arrest of tumor cells in circulation. Platelets interact with certain tumor cell emboli that could prolong cell survival in the circulation (48). Platelet–tumor cell emboli can also induce downstream ischemic endothelial damage, which could potentially expose adhesive subendothelial matrix for tumor cell binding and arrest (49). In addition, sequestration of tumor cells by platelets can protect tumor cells from immunological host surveillance (50). As described above, tumor

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cell-induced platelet activation and aggregation predominantly require prior activation of the coagulation system and thrombin generation, although other mechanisms independent of thrombin have also been reported (51). The anti-metastatic effect of experimental thrombocytopenia (52) and anti-platelet drugs such as aspirin (53) and dipyridamole (54) provide strong support for a requirement of platelets in hematogenous metastasis. Glycoprotein (Gp) IIb/IIIa plays a central role in homotypic platelet aggregation and is also involved in the heterotypic adhesion of tumor cells to platelets. Inhibition of metastasis by the anti-GpIIb/IIIa monoclonal antibody 10E5, which inhibits binding of fibronectin to von Willebrand factor to platelet GpIIb/IIIa, was first reported by Karpatkin and colleagues (55). In our laboratory, using a Lewis lung carcinoma (LL2) model of experimental metastasis in rats, we examined the effect of GpIIb/IIIa blockade on lung seeding. In this study, we utilized a murine F(ab’)2 version of abciximab (7E3), since the human antibody cross reacts with rat, but not mouse, GpIIb/IIIa and αvß3. This treatment inhibited tumor cell-induced thrombocytopenia and significantly reduced experimental metastasis (56). In a second study, using the LL2 lung carcinoma cells, this time in mice, we examined the effect of a potent non-peptide oral GpIIb/IIIa antagonist, XV454, on tumor cell-induced thrombocytopenia and experimental metastasis (57). XV454 has a long receptor-bound lifetime, similar to 7E3, and binds with high affinity to either activated or non-activated human, baboon, mouse or canine platelets (Kd = 0.5 nM) (58). Maximal aggregation inhibition by XV454 occurs at approximately 75% receptor occupancy. XV454 (5 mg/kg) was administered orally or intravenously 3 h or 10 min prior to tumor cell injection, respectively. By whole blood platelet aggregometry, we verified that mouse platelet aggregation was completely inhibited 1 h after oral and 10 min after intravenous administration of XV454. This inhibitory effect persisted for at least 24 h after oral delivery. Tumor cell-induced thrombocytopenia was inhibited significantly by both oral and intravenous administration of XV454. Similarly, both oral and intravenous XV454-treated mice had >80% fewer surface lung tumor nodules than the control group (P <0.001, n = 8 per group). However, tumor burden was reduced by 83% in animals receiving intravenous XV454 (P = 0.06), by 50% in the oral (single pre-tumor cell treatment) group (P = 0.14), and by 91% in the multiple treatment oral group (P = 0.015). Taken together, the results strongly suggest that platelet activation and aggregation are important for the success of metastases in these experimental models.

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4. Summary Our studies with an experimental model of tumor cell-induced coagulopathy and lung metastasis collectively suggest that TF is an important determinant of the clotting changes and the ability of tumor cells to form secondary tumors. Our data, and those of others, suggest that when procoagulant (TF-expressing) tumor cells enter the circulation, blood coagulation is rapidly activated, particularly at sites of tumor cell arrest. The rapid generation of thrombin leads to platelet activation and aggregation and these events both enhance the metastatic process. Because the primary trigger for these events is tumor cell TF, the ability to inhibit TFinduced coagulation, either downstream by reducing blood coagulability (heparins, coumadin) or more directly through anti-TF antibodies or small molecule TF inhibitors or via TFPI, may be a useful tool in the adjunct treatment of human malignancies. LMWHs may be particularly effective due to their dual effects of FXa inhibition and elevation of TFPI levels. However, despite preclinical evidence in favor of LMWH anti-tumor effects, clinical trials have reported conflicting results. A recent meta-analysis and systemic review of the efficacy and safety of anticoagulants (unfractionated heparin, LMWH, and warfarin) as cancer treatment concluded that these agents, particularly LMWH, significantly improved the overall survival of cancer patients without venous thrombosis (59). However, the authors also conclude that at the present time the use of anticoagulants as antineoplastic therapy cannot be recommended until more confirmatory randomized controlled trials are performed. Indeed, the translation of the above-described research into effective human therapy may require the development of novel anticoagulants with adequate bioavailability and safety profiles for their long-term use.

5. Notes 1. As mentioned above, the main disadvantage of animal models of experimental metastasis is the fact that large numbers of tumor cells are introduced directly into the blood stream. This is non-physiological and the initial steps of the metastatic process leading to intravasation are not considered. In addition, metastasis occurs as a result of the initial trapping of the tumor cells in the vasculature of the organ of

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first encounter (the lungs) and occurs very rapidly. Tumor cell-induced coagulation and platelet activation also occur more rapidly than in spontaneously metastasizing models and may be exaggerated by tumor cell trafficking in the microvessels. In our experience with several anticoagulant and anti-platelet intervention studies, using this model, antihemostatic agents only exert maximal anti-metastatic effect if administered before the injection of tumor cells. Indeed, we have found that delaying the administration of anticoagulant or anti-platelet agents significantly reduces their anti-metastatic effect. We believe that this is mainly due to the fact that the initial arrest and coagulation activation by tumor cells occur minutes after tumor cell injection and that anticoagulation after this time point does not significantly impact the metastatic process. 2. There are two alternatives to the intravenous models of experimental metastasis. The first model is known as a “spontaneous” model of metastasis. In these models tumor cells are injected subcutaneously into experimental animals and metastasis to the lung, liver, or other organs is assessed at various time points afterward. Although this model represents more closely the metastatic process, formation of distant tumor foci occurs very inefficiently. Often times the primary tumor grows to an inappropriately large volume without any visible signs of metastatic spread. The removal of the primary tumor has been shown to enhance metastatic spread. The second alternative is the use of orthotopic mouse models. In this model, tumor cells (or tumor fragments) are introduced into their organ of origin. The main advantage of this type of model is that theoretically at least, the growing tumor will follow its natural course of progression. In this regard, orthotopic models of tumor growth and dissemination represent human disease more closely than any other model. However, large variations in tumor growth patterns present difficulties in metastasis outcomes and data interpretation. 3. There are large variations in the number of pulmonary lung nodules in models of experimental metastasis. In our experience, in control groups we observe pulmonary nodules in 100% of injected control animals. However, there are large variations (e.g., range from 10 to 150) in the number of surface tumor foci. For this reason a minimum of 8–10 animals per experimental group is needed to obtain sufficient statistical rigor. 4. In some experimental metastasis models like the LL2 model, there are large variations in the sizes of the pulmonary tumor nodules. In addition, it is possible for multiple surface foci

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to coalesce and form apparently much larger nodules. When counting the surface nodules, it is recommended to consider small and large nodules similarly regardless of gross size differences. However, on these occasions, the measurement of tumor burden by weighing the entire lung tissue provides an additional and sometimes a more representative parameter for the evaluation of the anti-metastatic effects of potential therapeutic agents. 5. Experimental mice tolerate high daily doses of heparins and anti-GpIIb/IIIa agents (as highest used in our studies, 10–15 mg/kg) without any signs of bleeding. It is important to note that this tolerance can not be extrapolated to human studies. In humans, the therapeutic window of anticoagulants is certainly narrower than in rodents. This may be one reason why preclinical animal data on the anti-tumor effects of anticoagulants are more encouraging than those seen in human trials so far. References 1. De Cicco, M. (2004) The prothrombotic state in cancer: pathogenic mechanisms Crit Rev Oncol Hematol 50, 187–96. 2. Hillen, H.F. (2000) Thrombosis in cancer patients Ann Oncol (Suppl. 3) 11, 273–76. 3. Buller, H.R., van Doormaal, F.F., van Sluis, G.L., and Kamphuisen, P.W. (2007) Cancer and thrombosis: from molecular mechanisms to clinical presentations J Thromb Haemost 5(Suppl.1), 246–54. 4. Trousseau, A. Phlegmasia alba dolens. In: Baillier, J., ed. Clinique de l’Hotel-Dieu de Paris, 2nd edition. London: New Sydenham Society;1865:654–6. 5. Varki, A. (2007) Trousseau’s syndrome: multiple definitions and multiple mechanisms Blood 110, 723–9. 6. Langer, F., Spath, B., Haubold, K., et al. (2008) Tissue factor procoagulant activity of plasma microparticles in patients with cancerassociated disseminated intravascular coagulation Ann Haematol 87, 451–7. 7. Sun, N., McAfee, W., Hum, G., and Weiner J.M. (1979) Hemostatic abnormalities in malignancy, a prospective study of one hundred eight patients. Part 1. Coagulation studies Am J Clin Pathol 71, 10–6. 8. Kies, M.S., Posch, J.J., Giolama, J.P., and Rubin, R.N. (1980) Hemostatic function in cancer patients Cancer 46, 831–7. 9. Edwards, R.L., Rickles, F.R., Moritz, T.E. et al. (1987) Abnormalities of blood coagulation tests in patients with cancer Am J Clin Pathol 88, 596–602.

10. Davis, R.B., Theologides, A., and Kennedy, B.J. (1969) Comparative studies of blood coagulation and platelet aggregation in patients with cancer and non-malignant diseases Ann Int Med 71, 67–80. 11. Francis, J.L., and Amirkhosravi, A. (2002) Effect of antihemostatic agents on experimental tumor dissemination Semin Thromb Hemost 28, 29–38. 12. Rickles, F.R., Edwards, R.L., Barb, C., and Cronlund, M. (1983) Abnormalities of blood coagulation in patients with cancer: fibrinopeptide A generation and tumor growth Cancer 51, 301–7. 13. Francis, J.L., Francis, D.A., and Gunathilagan, G.J. (1994) Assessment of hypercoagulability in patients with cancer using the sonoclot analyzer and thromboelastography Thromb Res 74, 335–46. 14. Wojtukiewicz, M.Z., Sierko, E., Klement, P., and Rak, J. (2001) The hemostatic system and angiogenesis in malignancy Neoplasia 3, 371–84. 15. Bogdanov, V.Y., Balasubramanian, V., Hathcock, J., et al. (2003) Alternatively spliced human tissue factor: a circulating, soluble, thrombogenic protein Nat Med 9, 458–62. 16. Giesen, P.L., Rauch, U., Bohrmann, B., et al. (1999) Blood borne tissue factor: another view of thrombosis Proc Natl Acad Sci USA 96, 2311–5. 17. Hron, G., Kollars, M., Weber, H., et al. (2007) Tissue factor positive microparticles: cellular origin and association with

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Assessment of Anti-Metastatic Effects of Anticoagulant and Antiplatelet Agents 42. Snyder, L.A., Rudnick, K.A., Tawadros, R., et al. (2007) Expression of human tissue factor under the control of the mouse tissue factor promoter mediates normal hemostasis in knock-in mice J Thromb Haemost 6, 306–14. 43. Gemmell, C.H., Broze, G.J., Turitto, V.T., and Nemerson, Y. (1990) Utilization of a continuous flow reactor to study the lipoprotein–associated coagulation inhibitor (LACI) that inhibits tissue factor Blood 76, 2266–71. 44. Bajaj, M.S., Birktoft, J.J., Steer, S.A., and Bajaj, S.P. (2001) Structure and biology of tissue factor pathway inhibitor Thromb Haemost, 86, 959–72. 45. Rapp, J.H., Pan, X.M., Ghermay, A., et al. (1997) A blinded trial of local recombinant tissue factor pathway inhibitor versus either local or systemic heparin in a vein bypass model J Vasc Surg 25, 726–9. 46. Amirkhosravi, A., Meyer, T., Chang, J.Y., et al. (2002) Tissue factor pathway inhibitor reduces experimental lung metastasis of B16 melanoma Thromb Haemost 87, 930–6. 47. Fidler, I.J. (1970) Metastasis: quantitative analysis of distribution and fate of tumor emboli labeled with 125 I-5 deoxyuridine J Natl Cancer Inst 45, 773–82. 48. Hembrough, T.A., Swartz, G.M., Papathanassiu, A., et al. (2003) Tissue factor/factor VIIa inhibitors block angiogenesis and tumor growth through a nonhemostatic mechanism Cancer Res 63, 2997–3000. 49. Warren, B.A. The micro-injury hypothesis and metastasis. In: Honn, K.V., and Sloane, B.F., eds. Hemostatic Mechanisms and Metastasis.Boston: M. Nijhoff;1984:56. 50. Nieswandt. B., Hafner, M., Echtenacher, B., et al. (1999) Lysis of tumor cells by natural killer cells in mice is impeded by platelets Cancer Res 59, 1295. 51. Lerner, W.A., Pearlstein, E., Ambrogio, C., et al. (1983) A new mechanism for tumor-

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Chapter 11 Adhesion Molecules: Potential Therapeutic and Diagnostic Implications Shaker A. Mousa Abstract The role of cell adhesion molecules (CAMs) and extracellular matrix (ECM) proteins in various pathological processes, including angiogenesis, thrombosis, inflammation, apoptosis, cell migration, and proliferation is well documented. These processes can lead to both acute and chronic disease states such as ocular diseases, metastasis, unstable angina, myocardial infarction, stroke, osteoporosis, a wide range of inflammatory diseases, vascular remodeling, and neurodegenerative disorders. A key success in this field was identification of the role of platelet glycoprotein (GP)IIb/IIIa in the prevention and diagnosis of various thromboembolic disorders. The use of soluble adhesion molecules as potential diagnostic markers for acute and chronic leukocyte, platelet, and endothelial cell insult is becoming increasingly common. The development of various therapeutic and diagnostic candidates based on the key role of CAMs, with special emphasis on integrins in various diseases, as well as the structure–function aspects of cell adhesion and signaling of the different CAMs and ECM are highlighted. Key words: Integrins, selectins, immunoglobulins, CAM inhibitors, extracellular matrix proteins, αIIbβ3, αvβ3, αvβ5, α4β1, α4β7, α5β1, ICAM, VCAM, PECAM, soluble adhesion molecules, angiogenesis, apopotosis, thrombosis, restenosis, osteoporosis, inflammatory and immune disorders.

1. Introduction Many physiological processes, including cell activation, migration, proliferation, and differentiation require direct contact between cells or cells and extracellular matrix (ECM) proteins. Cell–cell and cell–matrix interactions are mediated through several different families of cell adhesion molecules (CAMs), which include selectins, integrins, cadherins, and immunoglobulins. The discovery of new CAMs, along with new roles for integrins, selectins, S.A. Mousa (ed.), Anticoagulants, Antiplatelets, and Thrombolytics, Methods in Molecular Biology 663, DOI 10.1007/978-1-60761-803-4_11, © Springer Science+Business Media, LLC 2003, 2010

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and immunoglobulins in certain disease states, provides great opportunities for the development of novel therapeutic and perhaps diagnostic modalities. Intensified drug discovery efforts directed at manipulating CAM activity through monoclonal antibodies, peptides, peptidomimetics, and non-peptide small molecules for diagnostic and therapeutic applications continue to broaden the scope of key clinical approaches. This chapter focuses on current advances in the discovery and development of novel anti-integrins for potential therapeutic and diagnostic applications, as well key methods of studying the different CAMs. CAMs play a very significant and critical role in both normal and pathophysiological disease states. For this key reason, the selection of specific and relevant CAMs to target certain disease conditions without interfering with other normal cellular functions is an important prerequisite for the ultimate success of an active and safe therapeutic strategy (1, 2). Exciting advances in our understanding of several CAMs, notably αvβ3, αvβ5, α4β1, α5β1, and αIIb/β3 integrin receptors and their direct relationships to different disease states open the door to tremendous therapeutic and diagnostic opportunities (1–7). Specific CAMs have been implicated in several different disease states, including cardiovascular disease and cancer, and are involved in a number of biological systems, including the inflammatory, ocular, pulmonary, bone, central nervous system, kidney, and gastrointestinal systems. The role of integrin αIIbβ3 in the prevention, treatment and diagnosis of various thromboembolic disorders is excellent proof of this concept (3–7). In addition, the potential prophylactic role of anti-selectins, the role of β1 and other leukointegrins in various inflammatory conditions, the potential utility of soluble adhesion molecules as surrogate markers for acute and chronic endothelial injury, and the potential role of αvβ3 in angiogenesis and osteoporosis have been reported (8, 9). The following sections will describe in more detail the therapeutic and diagnostic applications of selected CAMs.

2. Selectins and Anti-selectins Selectins comprise a family of three types of cell adhesion receptors (E-, L-, and P-selectins) that share common structural features, namely a lectin (L), EGF-like (E), and complement (C) binding-like domain (also termed LEC-CAMs). Functionally, selectins all mediate cellular interactions through the lectin domain of the selectin and cell surface carbohydrate ligands (10). P- and E-selections are calcium-dependent and are expressed

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on platelets or endothelial cells, where they mediate leukocyte adhesion through recognition of cell-specific carbohydrate ligands. L-selectins are found on all leukocytes and bind to their cognate receptor, Gly-CAM-1, a mucin-like endothelial glycoprotein (GP), on endothelial cells (11). E-selectin is an endothelial adhesion molecule whose expression is induced by various inflammatory stimuli. P-selectin is stored in alpha granules of platelets, as well as Weible-Palade bodies of endothelial cells. E-selectin recognizes the cell surface carbohydrate sialyl lewis x (SLeX) (12), while P-selectin recognizes a carbohydrate moiety that is closely related to SLeX (13). The selectin family of CAMs plays a key role in early neutrophil (PMN) rolling and adherence to endothelial cells (ECs). P-selectin on platelet and EC surfaces acts in concert with L-selectin on the leukocyte surface to promote PMN–EC and PMN–platelet interactions. Neutralizing monoclonal antibodies directed against P- or L-selectin preserve endothelial and monocyte cell function in an experimental model of myocardial ischemia/reperfusion injury (14, 15). In P-selectin deficient mice, P-selectin has been shown to play a role in neointima formation and potentially impact restenosis (16). L-selectin has also been shown to mediate PMN rolling interactions at sites of inflammation (17). 2.1. Assay for Human Soluble Selectin (sP-, sL-or sE-selectin)

2.1.1. Procedure

The mostly commonly used assay is a quantitative sandwich immunoassay technique. A microplate is pre-coated with a monoclonal antibody specific for sP-, sL- or sE-selectin. Standards, samples, and controls are dispensed into individual wells, together with a horseradish peroxidase (HRP)-conjugated polyclonal antibody specific for sP-, sL- or sE-selectin. After removal of unbound HRP-conjugated secondary antibody, a substrate is added and color develops proportional to selectin concentration. 1. Dilute samples as follows: for serum or plasma, a dilution of 1:100 should be adequate; for cell culture supernatants, use a dilution of 1:25. 2. Remove unused microtiter plates from the frame in which they were supplied, and store in a sealed foil pouch with a silica gel sachet. 3. Add 100 μL of standard, diluted sample or diluted parameter control to wells in duplicate. 4. Cover the plate with a plate sealer and incubate at room temperature for 1 h. 5. Add 100 μL of HRP-conjugated anti-selectin to each well with sufficient force to ensure mixing. Conjugate is red colored to facilitate accuracy in dispensing.

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6. Cover the plate with a new plate sealer (provided by the manufacturer) and incubate at room temperature for 30 min. 7. Aspirate or decant contents from each well and wash with 400 μl of wash buffer per well. Repeat the process five times for a total of six washes. After the last wash, aspirate or decant the contents and remove any remaining wash buffer by tapping the inverted plate firmly on a clean paper towel. 8. Add 100 μl of substrate to each well. Cover the plate and incubate at room temperature for 30 min. 9. Add 100 μl of stop solution to each well. The stop solution should be added to the wells in the same order as the substrate. 10. Determine the optical density (OD) of each well within 30 min using a microtiter plate reader (photometer) set at 450 nm with a correction wavelength of 620 nm. If the wavelength correction facility is not available, scan plates at 450 nm and then separately at 620 nm. Subtract OD620 from OD450 .

3. Integrins Integrins are a widely expressed family of cell adhesion receptors through which cells attach to the ECM, to each other, or to heterologous cells. All integrins are heterodimers composed of an α and β subunit. They are expressed on a wide variety of cells, and most cells express several different integrins. The interaction of integrins with the cytoskeleton and ECM appears to require the presence of both subunits. The binding of integrins to their ligands is cation dependent. Integrins recognize specific amino acid sequence motifs, the most well characterized of which is the RGD sequence found within a number of matrix proteins, including fibrinogen, vitronectin, fibronectin, thrombospondin, osteopontin, von Willebrand factor (vWF), and others. Integrins also bind to ligands via non-RGD sequences, such as the LDV sequence within the CS-1 region of fibronectin recognized by α4β1 integrin receptors. There are at least 8 known β subunits and 14 α subunits (1, 2). Integrin receptors contain an extracellular domain that engages adhesive ligands and a cytoplasmic face that engages intracellular proteins. Both of these interactions are critical for cell adhesion and anchorage-dependent signal transduction in normal and pathological states. For example, platelet activation induces a

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confirmational change in integrin αIIb/β3, thereby converting it into a high affinity fibrinogen receptor. Fibrinogen binding triggers a cascade of protein tyrosine phosphorylation and dephosphorylation through specific kinases and phosphatases, resulting in the recruitment of numerous other signaling molecules into macromolecular F-actin-rich cytoskeletal complexes assembled proximal to the cytoplasmic tails of the αIIb and β3 subunits. These dynamic structures regulate platelet function by coordinating and integrating signals emanating from integrins and G protein-linked receptors. Studies of integrin mutations confirm that the cytoplasmic tail of integrin αIIbβ3 is involved in signal transduction through direct interactions with cytoskeletal and signaling molecules. In terms of clinical relevance, blocking fibrinogen binding to the extracellular domain of integrin aIIbβ3 has been shown to effectively prevent the formation of platelet-rich arterial thrombi after coronary angioplasty (18). Once the full cohort of proteins that interact with the cytoplasmic tails of integrin αIIbβ3 are identified, it may be possible to develop selective inhibitors of integrin adhesion or signaling whose sites of action are intracellular. The commercial and therapeutic potential of CAMs is on the rise. The discovery of new CAMs, along with new roles for integrins, selectins, and immunoglobulins in certain disease states, opens the door to important opportunities in the development of therapeutic and diagnostic agents. Integrins represent one of the best opportunities for achieving small molecule antagonists for both therapeutic and diagnostic applications in several important diseases with unmet medical needs. 3.1. Potent and Selective Small-Molecule Antagonists of α 4 Integrins

The α4 integrins are central to leukocyte–cell and leukocyte– matrix adhesive interactions. Integrin α4β7 expression is restricted to leukocytes, with the exception of neutrophils. It interacts with the immunoglobulin superfamily member vascular-cell adhesion molecule 1 (VCAM-1), and an alternately spliced form of fibronectin (FN), as well as mucosal addressin-cell adhesion molecule 1 (MAdCAM-1), a mucosal vascular addressin, or homing receptor, that contains immunoglobulin-like domains related to VCAM-1 (19). Monoclonal antibodies to the α4 subunit or α4β7 can block the adhesive function of α4 and/or α4β7 integrins in vitro. Studies in vivo with these monoclonal antibodies in several species demonstrate that the interactions mediated by α4 integrins play key pathophysiological roles in immune and inflammatory reactions. Thus, α4 integrin-dependent adhesive interactions with VCAM-1, MAdCAM, and FN appear to play a central role in the recruitment, priming, activation, and apoptosis of certain leukocyte subsets. As such, α4 integrins represent novel targets for drug intervention. A selective and potent anti-α4 monoclonal antibody and small-molecule antagonists have shown in vivo

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efficacy in several experimental animal models (20, 21). MAdCAM-1 is an immunoglobulin-like adhesion receptor that is preferentially expressed by venular endothelial cells, defining sites of lymphocyte extravasation in mucosal lymphoid tissues and lamina popria. Peptide-based analogs based on various regions in the first and second immunoglobulin domains of MAdCAM-1 that mimic the binding of α4β7 have been identified. 3.2. β 1 Integrins

The β1 integrins, also known as the very late activation antigen (VLA) subfamily due to the late appearance of VLA after activation, comprise the largest subfamily of integrins. There are at least seven receptors of this subfamily, each with a different ligand specificity. Among the most well characterized are α4β1, α5β1, α6β1, and αnβ1 receptors. The leukocyte integrin α4β1 (also known as VLA-4 and CD49d/CD29) is a cell adhesion receptor expressed predominantly on lymphocytes, monocytes, and eosinophils (22). VLA-4 has been suggested as a potential target for therapeutics in chronic inflammatory diseases.

3.2.1. Leukocyte Integrin α 4β 1 as a Potential Therapeutic Target

Leukocyte populations that express α4β1 primarily mediate chronic inflammatory diseases (i.e., rheumatoid arthritis, asthma, psoriasis, and allergy). In contrast, VLA-4 is not present on circulating unstimulated neutrophils, which constitute a first line of defense against acute infections. Eosinophils selectively accumulate at sites of pulmonary inflammation in chronic allergic diseases such as bronchial asthma. The role of β1 integrins and their regulation by cytokines and other inflammatory mediators during eosinophil adhesion to the endothelium and ECM and during transendothelial migration has been well documented (23, 24). Interactions of VLA-4 with alternatively spliced forms of fibronectin containing the CS-1 region have been exploited in the design of small molecule inhibitors that bind to VLA-4 and block receptor function. Evaluation of these analogs in animal models of disease indicates that VLA-4 receptor blockers have the potential to achieve dramatic in vivo results in a variety of chronic inflammatory disorders (20–22). Infiltration of circulating immune cells into the central nervous system (CNS) can result in edema, myelin damage, and paralysis (25). Importantly, a role for integrin α4β1 in this infiltration process has been demonstrated. When administered to animals with experimental autoimmune encephalomyelitis (EAE), antibodies against α4 integrin prevent the adhesion of lymphocytes and monocytes to inflamed endothelia within blood vessels of the CNS and prevent immune cell infiltration. Even when administered to animals after the onset of paralysis, anti-α4

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antibodies reversed all clinical signs of disease. Magnetic resonance imaging analysis of these animals showed that antibody treatment reduced edema and reduced the permeability of the blood brain barrier to gadnolium-DPTA, and histological analysis demonstrated that antibody treatment prevented the destruction of myelin. Remarkably, anti-α4 antibodies reversed the accumulation of lymphocytes and monocytes within the CNS while having no affect on the level of these cells in circulation. These results suggest that the active disease process in EAE requires ongoing recruitment of circulating cells into the CNS and that antibodies directed against α4 integrins prevent this recruitment and reverse disease progression. 3.2.2. β 1 Integrins in Gastrointestinal (GI) Disease

Inflammatory bowel disease (IBD), Crohn’s disease, and ulcerative colitis (UC) are immunologically-based illnesses. The pattern of expression of β integrins in isolated intestinal lamina mononuclear cells from IBD and normal intestines is similar to that of normal solid organs. Isolated CD3+ cells from patients with Crohn’s disease express more β1 than normal individuals, supporting the idea that there are distinct β integrin systems involved in GI diseases. Interest in β1 and β7 integrins in particular as potential therapeutic targets for GI inflammatory disease remains high (26).

3.2.3. α 5β 1 Integrin in Angiogenesis and Bacterial Infection

Recent evidence suggests that α5β1 integrin is involved in the modulation of angiogenesis (27), suggesting that α5β1 antagonists might be useful in various angiogenesis-mediated disorders (27). Similarly, α5β1 integrin has been implicated in mediating bacterial invasion into human host cells leading to antibiotic resistance (28).

3.3. β 2 Integrins

The leukocyte-restricted β2 (CD18) integrins promote a variety of homotypic and heterotypic cell adhesion events required for normal and pathologic functioning of the immune system (2). Several physiological processes, including cell adhesion, activation, migration, and transmigration, require direct contact between cells or ECM proteins via CAM receptors. To date, three members of this integrin subfamily have been identified: CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), and CD11c/CD18 (P150,95). A fourth α chain, designated αd, has been cloned and shown to associate with CD18 in normal leukocytes and upon co-transfection into CHO cells. In vitro studies have demonstrated that LFA-1 and Mac-1 on neutrophils can be differentially activated, with distinct functional consequences (2). Studies in CD11b-deficient mice further underscore the biologic significance of distinct contributions of LFA-1 and Mac-1 to neutrophil-dependent tissue injury.

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3.4. α IIb/β 3 Integrin 3.4.1. Intravenous Platelet α IIb/β 3 Receptor Antagonists – Potential Clinical Utility

The realization that platelet integrin αIIbβ3 is the final common pathway for platelet aggregation regardless of the activation mechanism has prompted the development of several smallmolecule αIIbβ3 receptor antagonists for intravenous and/or oral anti-thrombotic use. Platelet αIIbβ3 receptor blockade represents a very promising therapeutic and diagnostic strategy for treating thromboembolic disorders. Clinical experience (i.e., studies of efficacy/safety) gained with injectable αIIbβ3 antagonists promises to yield valuable insight into the potential of longterm chronic use of oral αIIb/β3 antagonists. At this point, there are still many unanswered questions, however, and careful study will be needed to determine the safety and efficacy of this approach, either alone or in combination with other antiplatelet/anticoagulant therapies. Abciximab. The clinical utility of abciximab (ReoPro, c7E3 Fab) has been demonstrated in several trials involving coronary artery intervention procedures (29–31). The potent, rapid, and sustained block of platelet GPIIb/IIIa receptors, and perhaps αvβ3 as well, might be a key component of abciximab’s ability to mediate dramatic early anti-thrombotic benefits. Early benefits were maintained for over 3 years in patients receiving 12-h abciximab treatment in the EPIC (Evaluation of 7E3 for the Prevention of Ischemic Complications) trial. Integrilin. The IMPACT II (coronary intervention; broad entry criteria) and PURSUIT (unstable angina; chest pain <24 h, ischemic ECG changes) trials both demonstrated significant clinical benefits of integrilin, a cyclic heptapeptide KGD analog (32). Tirofiban. The RESTORE (coronary intervention; high risk of abrupt closure as per clinical and anatomic criteria), PRISM, and PRISM-plus (unstable angina; chest pain at 24 and 12 h) trials have demonstrated significant clinical benefits of tirofiban. Lamifiban. The PARAGON trial demonstrated significant clinical benefits of lamifiban (33). However, studies with lamifiban in Canada were stopped due to lack of efficacy and nuisance bleeding.

3.4.2. Chronic Therapy with Platelet GPIIb/IIIa Antagonists

When used for acute therapy of coronary arterial disease, GPIIb/IIIa antagonists must exhibit a high degree of platelet antagonism. By comparison, the requirements for chronic therapy using orally active agents have only recently been investigated. Interactions of oral GPIIb/IIIa antagonists with aspirin and other antiplatelet and anticoagulant drugs lead to shifts in the dose response curves for both efficacy and unwanted side effects, such as increased bleeding time (34–36). More recently, xemilofiban (EXCITE trial) and orbofiban (OUPIS trial), spon-

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sored by Searle, and sibrafiban, sponsored by Roche, were withdrawn because of disappointing outcomes. These results raise serious questions about the potential of oral GPIIb/IIIa antagonists (37, 38). Issues associated with oral GPIIb/IIIa antagonists currently under clinical investigation include thrombocytopenia, monitoring, bleeding risk, and drug interactions (39, 40). The role of the platelet integrin GPIIb/IIIa receptor and its potential utility as a radiodiagnostic agent for the rapid detection and/or diagnosis of thromboembolic events has been demonstrated (41). 3.5. α vβ 3 Integrin and Matrix Proteins in Vascular Remodeling

Processes involved in vascular remodeling also play a key role in the pathological mechanisms of atherosclerosis and restenosis. In response to vascular injury induced by percutaneous transluminal angioplasty (PTA), matrix proteins like osteopontin and vitronectin are rapidly up-regulated (42). Osteopontin stimulates smooth muscle cell migration through its action on integrin αvβ3 and thereby contributes to neointima formation and restenosis (43, 44). Osteopontin and vitronectin induce angiogenesis, which may also support neointima formation and arteriosclerosis (45). Thus, specific matrix proteins acting through integrin receptors, in particular, αvβ3, represent important targets for selective antagonists aimed at blocking the pathological processes of restenosis (42). Integrin αvβ3 ligands have also been utilized for the site-directed delivery of different therapeutic and diagnostic targets in oncology, in addition to having anti-cancer effects (46, 47).

4. Cellular and Integrin-Based Antiplatelet Efficacy Assays 4.1. Light Transmittance Aggregometry Assay

Venous blood is obtained from healthy non-smoker and nonfasted human donors (35–45 years of age, males and females) who have been drug- and aspirin-free for at least 2 weeks prior to blood collection (5, 6). Briefly, blood is collected into citrated Vacutainer tubes. The blood is subjected to centrifugation for 10 min at 150×g in a Sorvall RT6000 tabletop centrifuge equipped with a H-1000 B rotor at room temperature, after which platelet-rich plasma (PRP) is removed. After centrifugation of the remaining blood for 10 minutes at 1,500×g at room temperature, platelet-poor plasma (PPP) is removed. Samples are assayed on a PAP-4 Platelet Profiler, using PPP as the blank (set as 100% transmittance). PRP (200 μL at a concentration of 2×108 platelets/ml) is added to individual microcentrifuge tubes, and

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transmittance is set to 0%. The platelet agonist ADP (20 μL, 10 μM final concentration) is added to each tube and aggregation profiles are plotted as % transmittance over time. Antiplatelet agent (20 μl) is added at different concentrations ranging from 0.001–100 μM for 8 min prior to the addition of ADP. Results are expressed as percent inhibition of agonist-induced platelet aggregation, or IC50 (μM). 4.2. Platelet 125 I-Fibrinogen Binding Assay

Human PRP (h-PRP) is subjected to size exclusion chromatography to prepare human-gel-purified platelets (h-GPP). Aliquots of h-GPP (2×108 platelets/ml) along with 1 mM calcium chloride are added to removable 96-well plates. 125 I-Fibrinogen (26.5 μCi/mg) is added, and h-GPP are activated by the addition of ADP, epinephrine, and sodium arachidonate (100 μM each). 125 I-Fibrinogen bound to activated platelets is separated from the free form by centrifugation, and the radioactivity of the samples is counted using a gamma counter. Non-specific binding (due to entrapment of 125 I-fibrinogen) in the presence or absence of test agent (and in the absence of agonists) should be in the range of 4–6% of total 125 I-fibrinogen binding to agonist-activated platelets.

4.3. 293 β Fibrinogen Adhesion Assay

In this assay, αvβ3-transfected 293 cells are used. Adhesion of 293/β3 cells to fibrinogen is completely inhibited by an anti-αvβ3 monoclonal antibody, indicating that fibrinogen binding is dependent on integrin αvβ3. ELISA plates are coated with fibrinogen at a concentration of 25 μg/well and stored at 4ºC until use. On the day of the assay, plates are washed twice with phosphatebuffered saline (PBS) without cations, and the wells are incubated with 5% BSA/PBS for 2 h. 293/β3 cells at 30–70% confluence are harvested and resuspended at a density of 1×106 cells/ml. To each well of the 96-well plate, 65 μl of buffer is added, followed by 5 μl of different concentrations of test agent. Cells (130 μl) are added and the plates are incubated at 37ºC in 5% CO2 for 15 min. Non-adherent cells are removed, and remaining cells are lysed in a solution of 100 mM potassium phosphate, 0.2% Triton X-100, pH 7.8. An aliquot (5 ul) is assayed for β-galactosidase using a standard luminescence assay. Luminescence values are converted to β-galactosidase units using a standard curve, and data are normalized to correct for non-specific activity. Data are presented as percent inhibition and/or IC50 values.

4.4. SK-BR-3 Cell-Vitronectin (α vβ 5-Mediated) Adhesion Assay

An αvβ5-expressing breast cancer cell line (SK-BR-3; ATCC, Rockville, MD) is used for this assay. Adhesion of SK-BR-3 cells to vitronectin is αvβ5-dependent, as evidenced by complete inhibition of binding by an anti-αvβ5 monoclonal antibody (24). A Costar multi-well tissue culture plate is coated with 100 μl of vitronectin (0.25 μg per well) overnight at 4◦ C. The plate is

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washed twice with 200 μl of PBS, and then non-specific binding is blocked by incubating the plates with 200 μl of PBS+5% BSA for 1 h at room temperature. Cells are labeled with 2 μM calcein-AM (Molecular Probes) for 30 min at 37◦ C in a humidified incubator. Cells (1×106 cells/ml) are pre-incubated with 150 μl of test compound, or medium as a control, gently mixed, and then incubated for 15 min at room temperature. Treated cells are added to the assay plate in duplicate and then incubated for 60 min with shaking at room temperature. Plates are covered with foil to prevent photobleaching. Plates are washed to remove non-adherent cells, 100 μl of media is added to each well, and then fluorescence is measured using a Cytofluor 2300 system (excitation, 485 nM; emission, 530 nm). 4.5. Purified α 5β 1 ReceptorBiotinylated Fibronectin Binding Assay

Purified integrin α5β1 obtained from human placenta is coated onto Costar high capacity binding plates overnight at 4◦ C. The coating solution is discarded and plates are washed once with buffer. Wells are incubated with 200 μL of buffer containing 1% BSA. After washing once with buffer, 100 μl of biotinylated fibronectin (2 nM), plus 11 μl of test agent or buffer/BSA is added to each well and then the plates are incubated for 1 h at room temperature. Plates are washed twice with buffer and then incubated for 1 h at room temperature with 100 μl of alkaline phosphatase-conjugated anti-biotin antibody. Plates are washed twice with buffer and then incubated for 1 h with 100 μl of alkaline phosphatase substrate. Color is developed at room temperature for approximately 45 min, and then the reaction is stopped by the addition of 2 N NaOH. Plates are read at 405 nm. Functional assays of integrin-mediated intracellular signaling can be performed using transfected cells or other cell systems that express specific integrins and neutralizing or blocking antibodies or small molecule ligands specific for the integrin of interest. Integrin-mediated cell migration and/or proliferation can be studied using classical migration or proliferation assays.

5. Immunoglobulins Inter-cellular cell adhesion molecule (ICAM) and VCAM are members of the immunoglobulin (Ig) superfamily. At present, a great deal of effort in the targeting of Ig superfamily members is focused on the development of specific monoclonal antibodies and/or anti-sense oligonucleotides and small molecules that specifically block transcription factors. Strategies for the design of direct small molecular weight inhibitors of the Ig superfamily are somewhat more problematic. However, current advances in

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molecular modeling combined with advances in crystal structure data open the door to the development of cyclic peptides and peptidomimetic Ig antagonists. Monoclonal antibodies to ICAM-1 have anti-inflammatory properties, and show tremendous therapeutic potential in liver and kidney transplants, as well as in rheumatoid arthritis (48, 49). In contrast to current immunosuppressants, which are efficacious in organ transplant but have major adverse effects, anti-CAMs may prove to be more effective and safer. 5.1. Role of PECAM-1 in Regulating Transendothelial Migration of PMNs in Disease States

PMN’s adhere to the inflamed vascular endothelium, eventually undergoing transendothelial migration. The transmigration process is regulated largely by platelet/endothelial cell adhesion molecule-1 (PECAM-1), an adhesion receptor that is expressed on platelets, leukocytes and at the intercellular junctions of endothelial cells. PECAM-1 neutralizing antibodies selectively block PMN migration and markedly attenuate injury to ischemic-reperfused myocardium and coronary endothelium. Intravital microscopy demonstrated that the protective mechanism of PECAM-1 blockade involves inhibition of PMN transendothelial migration (50). Anti-PECAM-1 has been widely used as an indicator, typically in conjunction with immunostaining, of endothelial cells in various histological and molecular studies (51).

5.2. Soluble Adhesion Molecules as Surrogate Markers

CAMs are well recognized as adhesive receptors that facilitate adhesion, migration and transmigration of circulating cells into damaged vascular tissues. Recent studies have demonstrated that ICAM-1 is expressed on human athersclerotic plaques, and that treatment with an anti-ICAM-1 monoclonal antibody results in a significant reduction of myocardial infarct size in experimental myocardial/ischemia reperfusion injury models (55, 56). In addition, soluble isoforms of CAMs are believed to be shed from the surface of activated cells, and can be quantified in peripheral blood (52, 53). Increased serum concentrations of soluble CAMs have been documented in a variety of diseases (52, 53), suggesting the prognostic and diagnostic potential of various soluble adhesion molecules in vascular and cardiovascular disease.

5.3. Human Soluble (s)VCAM-1, ICAM-1, or PECAM Assay

The assay is based on the simultaneous binding of sVCAM-1 present in the sample or standard to two antibodies directed against different epitopes on the sVCAM-1 molecule. One antibody is adsorbed onto the surface of a microtiter well, and the other is conjugated to HRP. Any sVCAM-1 (or sICAM1 or PECAM) present will bridge the two different antibodies, allowing detection of the entire complex (antibody-sVCAM-1antibody). Briefly, after removal of unbound material by aspiration and washing, the amount of bound antibody complex

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is detected by reaction with an HRP substrate, which yields a colored product proportional to the amount of complex (and thus sVCAM-1) in the sample. The colored product can be quantified photometrically. By analyzing standards of known sVCAM1 concentration at the same time as the test samples, the concentration of the samples can be determined on a plot of signal versus concentration. 5.3.1. Procedure

For most samples (serum, plasma, or cell culture supernatant), a dilution of 1:50 should be adequate. 1. Add 100 μl of diluted anti-VCAM-1-HRP conjugate to each well. 2. Add 100 μl of standard, diluted sample, or diluted parameter control to each well with sufficient force to ensure mixing. Shaking or tapping is not recommended. 3. Cover the plate with a plate sealer and incubate at room temperature for 1.5 h. 4. Aspirate or decant contents from each well and wash by adding 300 μl of wash buffer per well. Repeat the process five times for a total of six washes. 5. After the last wash, aspirate or decant the contents and remove any remaining wash buffer by tapping the inverted plate firmly on clean paper toweling. 6. Immediately after decanting, add 100 μl of substrate to each well. 7. Cover the plate with a plate sealer and incubate at room temperature for 30 min. 8. Add 100 μl of stop solution to each well. The stop solution should be added to the wells in the same order as the substrate. 9. Determine the OD of each well within 30 min using a microtiter plate reader or Photometer set at 450 nm with a correction wavelength of 620 nm. The photometer or plate reader should be blanked according to the manufacturer’s instructions. If the wavelength correction function is not available, read plates at 450 nm and then again at 620 nm. Subtract OD620 from the OD450 .

6. Conclusion Several members of the different CAM superfamilies, in particular the integrin family, represent excellent potential therapeutic and diagnostic targets for a number of diseases with unmet medical

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needs. The identity of the CAM within a specific pathophysiological aspect of the disease process as well as the ease in achieving a small molecule anti-CAM candidate will ultimately determine the success of this particular class of therapeutic and diagnostic agent. References 1. Cox, D., Aoki, T., Seki, J., Motoyama, Y., and Yoshida, K. (1994) The pharmacology of the integrins Med Res Rev 14, 195–228. 2. Lal, H., Verma, S.K., Foster, D.M., Golden, H.B., Reneau, J.C., Watson, L.E., Singh, H., and Dostal, D.E. (2009) Integrins and proximal signaling mechanisms in cardiovascular disease Front Biosci 14, 2307–34. 3. Gold, H.K., Gimple, L.W., Yasuda, T., Leinbach, R.C., Werner, W., Holt, R., Jordan, R., Berger, H., Collen, D., and Coller, B.S. (1990) Pharmacodynamic study of F(ab’)2 fragments of murine monoclonal antibody 7E3 directed against human platelet glycoprotein IIb/IIIa in patients with unstable angina pectoris J Clin Invest 86, 651–9. 4. Mousa, S., and Topol, E. Novel antiplatelet therapies: recent advances in the development of platelet GPIIb/IIIa receptor antagonists. In: Serruys, P.W., and Holmes, D., eds. Current Review of Interventional Cardiology, 3rd edition. Philadelphia, PA: Current Medicine;1997:114–29. 5. Mousa, S.A., Bozarth, J.M., Forsythe, M.S., Jackson, S.M., Leamy, A., Diemer, M.M., Kapil, R.P., Knabb, R.M., Mayo, M.C., Pierce, S.K., et al. (1994) Antiplatelet and antithrombotic efficacy of DMP 728, a novel platelet GPIIb/IIIa receptor antagonist Circulation 89, 3–12. 6. Mousa, S.A., Bozarth, J.M., Forsythe, M.S., Lorelli, W., Thoolen, M.J., Ramachandran, N., Jackson, S., De Grado, W., and Reilly, T.M. (1993) Antiplatelet efficacy and specificity of DMP728, a novel platelet GPIIb/IIIa receptor antagonist Cardiology 83, 374–82. 7. van’t Hof, A.W., and Valgimigli, M. (2009) Defining the role of platelet glycoprotein receptor inhibitors in STEMI: focus on tirofiban Drugs 69, 85–100. 8. Brooks, P.C., Clark, R.A., and Cheresh, D.A. (1994) Requirement of vascular integrin alpha v beta 3 for angiogenesis Science 264, 569–71. 9. Brooks, P.C., Montgomery, A.M., Rosenfeld, M., Reisfeld, R.A., Hu, T., Klier, G., and Cheresh, D.A. (1994) Integrin alpha v beta 3 antagonists promote tumor regression by

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45. van der Zee, R., Murohara, T., Passeri, J., Kearney, M., Cheresh, D.A., and Isner, J.M. (1998) Reduced intimal thickening following alpha(v)beta3 blockade is associated with smooth muscle cell apoptosis Cell Adhes Commun 6, 371–9. 46. Kerr, J.S., Slee, A.M., and Mousa, S.A. (2002) The alpha v integrin antagonists as novel anticancer agents: an update Expert Opin Investig Drugs 11, 1765–74. 47. Oba, M., Fukushima, S., Kanayama, N., Aoyagi, K., Nishiyama, N., Koyama, H., and Kataoka, K. (2007) Cyclic RGD peptideconjugated polyplex micelles as a targetable gene delivery system directed to cells possessing alphavbeta3 and alphavbeta5 integrins Bioconjug Chem 18, 1415–23. 48. Flavin, T., Ivens, K., Rothlein, R., Faanes, R., Clayberger, C., Billingham, M., and Starnes, V.A. (1991) Monoclonal antibodies against intercellular adhesion molecule 1 prolong cardiac allograft survival in cynomolgus monkeys Transplant Proc 23, 533–4. 49. Haug, C.E., Colvin, R.B., Delmonico, F.L., Auchincloss, H., Jr., Tolkoff-Rubin, N., Preffer, F.I., Rothlein, R., Norris, S., Scharschmidt, L., and Cosimi, A.B. (1993) A phase I trial of immunosuppression with anti-ICAM-1 (CD54) mAb in renal allograft recipients Transplantation 55, 766–72; Discussion 72–3. 50. Rosenblum, W.I., Nelson, G.H., Wormley, B., Werner, P., Wang, J., and Shih, C.C. (1996) Role of platelet-endothelial cell adhesion molecule (PECAM) in platelet adhesion/aggregation over injured but not denuded endothelium in vivo and ex vivo Stroke 27, 709–11. 51. Muller, W.A., and Randolph, G.J. (1999) Migration of leukocytes across endothelium and beyond: molecules involved in the transmigration and fate of monocytes J Leukoc Biol 66, 698–704. 52. Gearing, A.J., and Newman, W. (1993) Circulating adhesion molecules in disease Immunol Today 14, 506–12. 53. Newman, W., Beall, L.D., Carson, C.W., Hunder, G.G., Graben, N., Randhawa, Z.I., Gopal, T.V., Wiener-Kronish, J., and Matthay, M.A. (1993) Soluble E-selectin is found in supernatants of activated endothelial cells and is elevated in the serum of patients with septic shock J Immunol 150, 644–54.

Chapter 12 Pharmacogenomics in Thrombosis Shaker A. Mousa Abstract Inherited or acquired genetic abnormalities play a major role in thromboembolic complications. The goal of pharmacogenomics is to tailor medications to an individual’s genetic makeup in order to improve the benefit-to-risk ratio. Significant findings have been documented showing the effect of certain genetic variations (e.g., in CYP2C9 and VKORC1) on the dose response to warfarin. Pharmacogenomic and genetic information is crucial to improving the efficacy and safety of pharmacotherapy and for the optimal management of thromboembolic disorders. Key words: Pharmacogenomics, cardiovascular disease, cytochrome P450, warfarin, single nucleotide polymorphisms.

1. Introduction According to the American Heart Association, approximately 80 million people (one out of three) have one or more forms of cardiovascular disease (CVD) (1). This number places a major burden on drug discovery efforts to improve the treatment of CVD. In today’s world of prescribed medications, doctors commonly engage in the practice of trial and error. Consider, however, the possibility of doctors being able to prescribe medications based on a patient’s specific genetic makeup, knowing in advance how the patient will respond (2, 3). This is the promise of pharmacogenomics. The term pharmacogenetics, first introduced by Vogel in 1959 (4), is defined as the analysis of inherited factors that define an individual’s response to a drug. The term generally refers to the identification and analysis of monogenetic variants that affect S.A. Mousa (ed.), Anticoagulants, Antiplatelets, and Thrombolytics, Methods in Molecular Biology 663, DOI 10.1007/978-1-60761-803-4_12, © Springer Science+Business Media, LLC 2003, 2010

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drug response. Pharmacogenomics, on the other hand, refers to the identification and analysis of the entire library of genes that impact drug efficacy and safety. The most common genetic variations are single nucleotide polymorphisms (SNPs), which occur on average at least once every 1,000 base pairs. SNPs account for nearly 3 million genetic variations and are distributed throughout the entire genome. Genetic variations that occur at a frequency of at least 1% in the human population are referred to as polymorphisms. Genetic polymorphisms are inherited and are monogenic (i.e., involving one locus) and exhibit inter-ethnic differences in frequency. Rare mutations are those that occur at a frequency of less than 1% in the human population. Other examples of genetic variations include insertion–deletion polymorphisms, tandem repeats, defective splicing, aberrant splice sites, and premature stop codon polymorphisms. There are many different factors that will contribute to how a patient responds to certain medications, such as age, gender, body weight, nutrition status, organ function status, presence of infections, and concomitant medications. We can now add genetic factors to this list (5). In addition, there has been a recent shift from looking at single genes (genetics) to focusing on the function and interactions of the whole genome (6). A major focus of current research in the field of pharmacogenomics is on cardiovascular medicine (7). The goal of pharmacogenomics is personalized medicine, in which the type of drug and dosing regimen are tailored for each individual, as opposed to the one-drug-fits-all approach of most current medical practices. An important facet of the pharmacogenomic approach is being able to predict who will respond to a specific drug and who will experience adverse reactions (8). Even with the most advanced medications, not every patient has a full response to every drug (9). A significant clinical problem with current cardiovascular medications is adverse drug reactions, which are a major cause of hospitalization in the United States today. Pharmacogenomics could help identify patients that will experience adverse effects, thus avoiding potential toxicity and even death (8). It is generally believed that individualized therapy based on pharmacogenomics will result in more effective, safer medications and more accurate dosing regimens, both of which can lead to decreased health-care costs and improved costeffectiveness by reducing hospitalizations due to adverse events, and decreasing the number of failed drug attempts in an effort to find and establish an effective regimen (7). With advances in genotyping technologies, we are now well on our way to being able to understand how genetic variations can impact treatment (8). The precise mechanism(s) underlying the variability in drug response among individuals is not clear; however, there is considerable evidence that genetic makeup is

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at least partially responsible (10). Most research in this area has focused on SNPs and DNA copy number variants (CNVs). The goal of such studies is to link SNPs or CNVs to the expression of a target gene, which can be assessed using DNA microarrays, and thereby determine how an individual will respond to a given medication (8). One of the current challenges of pharmacogenomics is that while we know that both genetic and non-genetic factors can influence an individual’s response to a particular drug, and these factors are beginning to be identified, it is not clear the degree to which each factor effects variations in drug response (8). One area of pharmacogenomics that has been incorporated into current clinical practice is genetic variation in the genes for the cytochrome P450 (CYP) enzymes. CYP enzymes metabolize many classes of medications, including cardiovascular drugs. Variations in the genes that encode CYP enzymes can result in the synthesis of overactive or inactive forms of the enzymes (see below). Patients who carry an inactive enzyme variant will be unable to metabolize and eliminate drugs properly, resulting in drug accumulation and possible serious toxicity (5). The inclusion of pharmacogenetic data on CYP polymorphisms has begun to be included in the packaging information that accompanies certain drugs. The package insert for warfarin, for example, provides physicians with genomic information related to drug dosing and how individual responses to the drug may vary (11).

2. SNP Mapping With the completion of the human genome, gene-based approaches using SNP markers have become an important tool in the identification of underlying causes, diagnosis, and treatment of disease. Through linkage disequilibrium (LD) analysis, or analysis of non-random association between SNPs in proximity to each other, tens of thousands of anonymous SNPs can be identified and mapped. These anonymous SNPs may fall within non-related genes, within susceptibility genes, or in non-coding sequences. Once identified and mapped, SNP markers can be used to identify a region of the genome that harbors a putative “susceptibility gene,” i.e., genetic variations that directly influence the likelihood that an individual will develop disease. Positional cloning and sequence analysis then identifies the gene and/or the SNP that underlies a specific condition or disease (12). The concept of susceptibility genes has led to the identification of a number of putative gene variants associated with hematologic, haemostatic, and thrombotic disorders (Table 12.1). Notably, variations in the gene for thiopurine methyltransferase

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Table 12.1 Genes involved in hematologic, haemostatic, and thrombotic disorders Clone ID

Name

Description

810512

THBS1

Thrombospondin 1

205185

THBD

Thrombomodulin

143443

TBXAS1

Thromboxane A synthase 1

812276

SNCA

Synuclein, alpha (non-A4 component of amyloid precursor)

149910

SELL

Selectin E (endothelial adhesion molecule 1)

753211

PTGER3

Prostaglandin E receptor 3 (subtype EP3)

245242

CPB2

Carboxypeptidase B2 (plasma, carboxypeptidase U)

130541

PECAM1

Platelet/endothelial cell adhesion molecule (CD31 antigen)

40643

PDGFRB

Platelet-derived growth factor receptor, beta polypeptide

121218

PF4

Platelet factor 4

66982

PLGL

Plasminogen like

810017

PLAUR

Plasminogen activator, urokinase receptor

813841

PLAT

Plasminogen activator, tissue serine (or cystein) proteinase inhibitor, Clade E (nexin, plasminogen activator inhibitor type 1), member 1

160723

LAMC1

Laminin, gamma 1 (formerly LAMB2)

32609

LAMA4

Laminin, alpha 4

51447

FCGR3B

Fc fragment of IgG, low-affinity IIIb, receptor for Z(CD16)

41898

PTGDS

Prostaglandin D2 synthase (21kD, brain)

810124

PAFAH1B3

Platelet activating factor acetylhydrolase, isoform 1b, gamma subunit (29kD)

810010

PDGFRL

Platelet-derived growth factor receptor like

184038

SPTBN2

Spectrin, beta, non-erythrocytic 2

179276

FASN

Fatty acid synthase

776636

BHMT

Betaine-homocystein methyltransferase

770462

CPZ

Carboxypeptidase Z

137836

PDCD10

Programmed cell death 10

127928

HBP1

HMG-box containing protein 1

138991

COL6A3

Collagen, type VI, alpha 3

212649

HRG

Histidine-rich glycoprotein

155287

HSPA1A

Heat shock 70 kD protein 1A

810891

LAMA5

Laminin, alpha 5

811792

GSS

Glutathione synthetase

768246

G6PD

Glucose-6-phosphate dehydrogenase

260325

ALB

Albumin

131839

FOLR1

Folate receptor 1 (adult)

139009

FN1

Fibronectin 1

813757

FOLR2

Folate receptor 2 (fetal)

241788

FGB

Fibrinogen, B beta polypeptide

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Table 12.1 (continued) Clone ID

Name

Description

49509

EPOR

Erythropoietin receptor

26418

EDG1

Endothelial differentiation, sphingolipid G-protein-coupled receptor.

1813254

F2R

Coagulation factor II (thrombin) receptor

261519

TNFRSF5

Tumor necrosis factor receptor superfamily, member 5

243816

CD36

CD36 antigen (collagen type 1 receptor, thrombospondin receptor)

714106

PLAU

Plasminogen activator, urokinase

758266

THBS4

Thrombospondin 4

712641

PRG4

Proteoglycan 4 (megakaryocyte stimulating factor, articular superficial zone protein)

726086

TFPI2

Tissue factor pathway inhibitor 2

67654

PDGFB

Platelet-derived growth factor beta polypeptide (simian sarcoma viral (v-sis) oncogene homolog

85979

PLG

Plasminogen

85678

F2

Coagulation factor II

814378

SPINT2

Serine protease inhibitor, kunitz type 2

71101

PROCR

Protein C receptor, endothelial (EPCR)

785975

F13A1

Coagulation factor XIII, A1 polypeptide

310519

F10

Coagulation factor X

840486

VWF

Von Willebrand factor

191664

THBS2

Thrombospondin 2

(TPMT), a drug metabolizing enzyme, and 5-lipoxygenase (ALOX5) have been linked to adverse drug reactions and variability in drug response, respectively (13, 14). An understanding of genetic variability is particularly important in the context of safety and efficacy of anticoagulant drugs. 2.1. SNPs in Drug Metabolizing Enzymes

Polymorphisms in drug metabolizing enzymes are the first recognized and most documented examples of genetic variations that have consequences not only in drug response but also drug toxicity. Drug metabolizing enzymes are divided into phase-I and phase-II metabolizing enzymes.

2.1.1. Phase-I Metabolizing Enzymes

Most phase-I enzymes are members of the CYP superfamily and localize to cells of the liver and gastrointestinal system. Approximately 40 different CYP enzymes are present in humans. They are classified according to family (e.g., 2), subfamily (e.g., D), and gene (e.g., 6) associated with the biotransformation (e.g., CYP2D6). Functional genetic polymorphisms have been identified for CYP2A6, CYP2C9, CYP2C19, and CYP2D6 (15),

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and more recently for CYP3A4 (16). A polymorphism in the regulatory region of the gene encoding CYP1A2 has been recognized, but its functional importance is unknown (17). CYP2CP, the primary pathway of metabolism of warfarin, metabolizes the S-isomer of the racemic warfarin mixture, which is 3 times more potent than the R-isomer. The two most common CYP2C9 variants, CYP2C9∗ 2 and CYP2C9∗ 3, have single amino acid substitutions at critical positions in the enzyme (18). CYP2C9∗ 3 homozygotes can exhibit as high as 90% reduction in S-warfarin clearance as compared to wild-type carriers (19). CYP2C9 mutant alleles were found to be overrepresented in 81% of patients requiring low-dose warfarin therapy (<1.5 mg/d) (20). These patients typically have difficulty in induction, require longer hospitalization for stabilization of the warfarin regimen and experience more bleeding complications. Patients that were homozygous for the CYP2C9∗ 3 allele had a profound response to warfarin, necessitating a dose reduction to 0.5 mg/d. It is now recognized that polymorphisms in the CYP genes can result in three possible drug phenotypes: poor, normal, and ultrafast metabolizers. Poor metabolizers lack an active form of the expressed enzyme, normals have one copy of the active gene, and ultrafast metabolizers have duplicate copies of the active gene. Poor metabolism results in toxicity (the drug stays in the body for a longer period of time), while ultrafast metabolism results in reduced drug efficacy. More than half of prescribed drugs are metabolized by components of the CYP system. Of this half, CYP3A4 metabolism accounts for approximately 50%, CYP2D6 accounts for 20%, and CYP2D9 and CYP2D19 account for 15%. In the future, drug developers will be able to identify and avoid drugs whose metabolic pathways are significantly influenced by genetic polymorphisms in the CYP system. 2.1.2. Phase-II Metabolizing Enzymes

Examples of phase-II metabolizing enzymes that exhibit genetic polymorphisms are N-acetyltransferase, TPMT, and glutathione S-transferase. The relevance of genetic polymorphisms of TPMT, dihydropyrimidine dehydrogenase (DPD) and UDPglucuronosyl transferase (UGT) in cancer have been demonstrated (21–23). The TPMT gene has three mutant alleles, referred to as TPMT∗ 3A, TPMT∗ 2, and TPMT∗ 3C, with the most common being TPMT∗ 3A.

2.2. Drug Transporter Gene Polymorphisms

Drug transport across the gastrointestinal lining, drug excretion into the bile and urine, and drug distribution across the blood– brain barrier are mediated by specific membrane proteins. Genetic variations in these proteins can cause disturbances in the distribution of a drug and alter drug concentrations, both of which will affect the therapeutic action and efficacy of the drug. The P-glycoprotein complex is an energy-dependent transmembrane

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efflux pump that is encoded by MDR-1 (multi-drug resistant gene 1). Fifteen MDR-1 polymorphisms have been identified to date. The most common, located in exon 26 of MDR-1 (24), has been shown to influence P-glycoprotein expression in vitro (24).

3. Pharmacogenomics of Warfarin Therapy

Some of the most promising pharmacogenomic data at present for CVD medications center on the anticoagulant warfarin. Warfarin is the most commonly prescribed anticoagulant for the prevention and treatment of thromboembolic events (25). However, warfarin is known to have a narrow therapeutic index, and inappropriately high doses can cause significant bleeding events. Thus, anticoagulation status in patients receiving warfarin must be monitored, particularly upon initiation of treatment, typically through evaluation of prothrombin time and international normalized ratio (INR) (26). Warfarin is metabolized via the hepatic CYP enzyme CYP2C9. Because of its narrow therapeutic index, warfarin is highly sensitive to drugs that inhibit or enhance CYP2C9 activity. In addition, genetic variations in CYP2C9 can cause some patients to metabolize warfarin more slowly, in which case the drug remains in the body for a longer period of time, putting the patient at an increased risk of serious bleeding (26). There are two main variant alleles of CYP2C9 (Table 12.2), referred to as the ∗ 2 allele and the ∗ 3 allele (25). These alleles have been shown to cause reductions in CYP2C9 enzymatic activity by approximately 30 and 80%, respectively, thus increasing the risk of warfarin-associated bleeding events (25). The mean warfarin dose required in patients with the ∗ 3 or ∗ 2 allele is significantly lower than in those patients without these alleles. These findings are highly suggestive of a gene–dose relationship between CYP2C9 and warfarin (27). Variant alleles are associated with an increased time to reach stable dosing and an increased risk of above range INRs (INR > 3.2) as compared to wild-type carriers (25, 26). Importantly, variant alleles are associated with an increased risk of bleeding events, with the ∗ 3 allele having a higher

Table 12.2 Polymorphisms associated with variable drug response to warfarin Gene

Polymorphisms

Functional role

Reference(s)

CYP2C9

∗ 2 and ∗ 3 alleles

VKORC1

Warfarin metabolism

(20, 29, 30, 32, 34, 45)

−1639G>A and 1173C>T

Activation of clotting factors (20, 29, 30, 32, 45)

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risk of bleeds (above range INRs) and a longer time to reach stable dosing as compared to the ∗ 2 allele. Ideally, the use of genotyping to guide dosing of warfarin will reduce the risk of above range INRs and bleeding events and reduce time to stable dosing (26). The identification of genetic variants of the gene for the warfarin target, vitamin K epoxide reductase (encoded by VKORC1), has revealed another putative pharmacodynamic mechanism of warfarin resistance. Warfarin inhibits the activity of VKOR, resulting in decreased levels of reduced vitamin K, which is required for the function of several clotting factors (Fig. 12.1). Genetic variations in VKORC1 that result in reduced activity of the enzyme, and thus decreased function of vitamin K-dependent clotting factors, have been described (28). Several VKORC1 SNPs have been identified, but two in particular, at position –1639 of the VKORC1 promoter and +1173 in intron 1 of VKORC1, were found to correlate significantly with warfarin dose requirements. For both of these positions, there was a significant risk of increased INRs with variant alleles (25, 27). Thus, in addition to such variables as age, sex, diet, and weight, genetic information about CYP2C9 and VKOR status may someday be considered in determining warfarin dosing regimes for individual patients (27).

Warfarin

CYP1A1 CYP1A2 CYP3A4

Swa rfa rin

n ari arf w R-

Vitamin K Reductase

Oxidized Vitamin K

CYP2C9

Reduced Vitamin K CO2 O2

Calumenin

Hypofunctional F. II, II, VII, VII,IX, IX,XX Protein C, S, Z

γ -glutamyl carboxylase

Functional F. II, II, VII, VII,IX, IX,XX Proteins C, S, Z

Fig. 12.1. Schematic representation of warfarin metabolism and the role of vitamin K reductase. The more potent S-warfarin is metabolized mainly via CYP2C9. Many clotting factors are dependent on the warfarin target, VKOR1. Polymorphisms in CYP2C9 or VKOR1 may account in large part for inter-patient variability in warfarin response.

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285

This section summarizes important genetic mutations that have been shown to be associated with CVD and hematological disorders. Additional potentially relevant polymorphisms associated with CVD, general metabolism, and immune system function are summarized in Table 12.3. 1. Coagulation factor II (prothrombin) G20210A. This mutation, present in 2% of the population, is located in the 3’ untranslated region, near a putative polyadenylation site. It is associated with increased levels of prothrombin, increased risk of deep vein thrombosis (DVT), recurrent miscarriages and portal vein thrombosis in cirrhotic patients (29–32). 2. Coagulation factor V G1691A (Leiden R506Q). The G1691A mutation, present in 8% of the population, is a specific G to A substitution at nucleotide +1691. The

Table 12.3 Polymorphisms associated with CVD, metabolism and immune function Gene

Polymorphism(s)

Association

Endothelial nitric oxide synthase (eNOS)

E298D, G894T

Increased risk for acute MI, coronary atherosclerosis, and essential hypertension

Coagulation factor II (Prothrombin)

G20210A

Elevated prothrombin levels with increased risk of DVT

Factor V (Leiden)

G1691A

Increased risk of venous thrombosis

Factor XIII

V34L, G103T

Lower incidence of CVD

Methylenetetrahydrofolate reductase (MTHFR)

C677T

Major risk factor for vascular disease

Apolipoprotein E

E2–E3–E4

General metabolism

Butyrylcholinesterase

atypic and K-variants

General metabolism

Leukotriene C4 Synthase

A-444c

General metabolism

IL1β

C4336T (TaqI RFLP)

Immune system and host immune response

IL4Rα

Q576R

Immune system and host immune response

IL6

G-174C

Immune system and host immune response

TNFα

G-238A, G-308A

Immune system and host immune response

TNFβ

A329G

Immune system and host immune response

CD14

C-260T

Immune system and host immune response

TLR4

Immune system and host immune response

CCR2-V641

(G190A)

Immune system and host immune response

SDF-1

3’ G810A

Immune system and host immune response

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Mousa

resultant gene product is cleaved less efficiently (10%) by activated protein C. The mutation is associated with DVT, recurrent miscarriage, portal vein thrombosis in cirrhotic patients, early kidney transplant loss, and other forms of venous thromboembolism (29–31, 33), as well as a dramatic increase in the incidence of thrombosis in women taking oral contraceptives. 3. Coagulation factor IX propeptide missense mutations. Alanine-10 to threonine and alanine-10 to valine missense mutations in the factor IX propeptide result in abnormal sensitivity to oral anticoagulants through reduced affinity of carboxylase for the vitamin K-dependent coagulation factor IX precursor, resulting in severe bleeding complications (“coumarin hypersensitivity”). Patients with coumarin hypersensitivity experience severe bleeding, despite a therapeutic INR. Increased activated partial thromboplastin time (aPTT) in these patients is due to reduced levels of factor IX. In the absence of coumarins, aPTT and factor IX levels are normal. Thus, analysis of nucleotide substitutions at position +9311 (G>A threonine variant) or position +9312 (C>T valine variant) are important in order to identify putative hypersensitivity before initiation of coumarin therapy to avoid excessive bleeding complications (34–37). 4. Platelet glycoprotein Ia C807T. This gene polymorphism is associated with nonfatal myocardial infarction in younger patients. Other platelet polymorphisms, such as those found in P-selectin, α2 adrenergic receptor, and transforming growth factor-β (TNF-β) are also associated with increased risk of arterial disease or a prothrombotic state (38–41). 5. Platelet glycoprotein IIIa T393C (HPA-1 a/b, P1A1/A2). The glycoprotein IIIa P1A1/A2 alleles are associated with a leucine 33/proline 33 amino acid polymorphism and are distinguishable by DNA typing and alloantibodies (42–44). HPA-1a (human platelet antigen-1a) was recently identified as an inherent risk factor for coronary thrombosis, premature myocardial infarction, and coronary stent thrombosis. HPA-1a is also a determinant in the pathogenesis of posttransfusion purpura and neonatal alloimmune thrombocytopenic purpura.

5. Summary and Future Directions Variability in drug response in different patients may be due to genetic differences that affect drug metabolism, drug distribution, and drug target proteins (15). Variations in CYP enzymes

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cause inter-individual differences in the plasma concentrations of drugs such as warfarin that have a narrow therapeutic index (20). In patients that experience adverse effects, medications may be either avoided or administered at a reduced dose with careful monitoring, neither of which is ideal. Increased knowledge of the regulation of cellular functions and drug effects at the genetic level should lead to the development of new drugs and/or drug combinations that are tailored to an individual’s needs. Through the discovery of new genetic targets, pharmacogenomics has the potential to improve quality of life and reduce health-care costs by decreasing the number of treatment failures and adverse drug reactions. Adverse drug reactions are the sixth leading cause of death in the United States, with an annual expenditure of $75 billion. In addition to age, sex, and nutritional status, genetic factors play a significant role in individual’s response to a drug. There are a number of inherited sequence variants (alleles) of genes that encode drug metabolizing enzymes and drug receptors that manifest as discrete drug metabolism phenotypes. Ideally, pretreatment screening for these alleles, thereby enabling predictions of a patient’s response to a medication, will help in selecting a drug and dosing regime that is safe and efficacious. The future may very well see physicians requesting a genotyping test instead of a complete blood count, for example, in order to diagnosis and prescribe an individualized treatment. In this regard, recent advances in molecular biological techniques for pin-pointing relevant mutations will be important. Pharmacogenomics-based personalized medicine holds the promise of reducing the number of adverse drug events and drug failures, thereby vastly improving quality of life while reducing health-care costs. Pharmacogenomics also has the potential to provide key insights for the diagnosis of drug resistance in thrombosis and beyond. References 1. Cardiovascular Disease Statistics. American Heart Association, 2009 (Accessed January 13, 2009, at http://www.americanheart. org/presenter.jhtml?identifier=4478). 2. Pharmacogenomics. Human Genome Project Information, 2008. (Accessed January, 2009, at http://www.ornl.gov/sci/techresources/ Human_Genome/medicine/pharma.shtml). 3. Aspinall, M., and Hamermesh, R. (2007) Realizing the promise of personalized medicine Harv Bus Rev 85, 108–17, 65. 4. Vogel, F. (1959) Moderne Probleme der Humangenetick. Ergebn Inn Med Klinderheilk 12, 52–125.

5. Sadee, W., and Dai, Z. (2005) Pharmacogenetics/genomics and personalized medicine Hum Mol Genet 14(Spec No. 2), R207–14. 6. Khoury, M. (2003) Genetics and genomics in practice: the continuum from genetic disease to genetic information in health and disease Genet Med 5, 261–8. 7. Ginsburg, G., Donahue, M., and Newby, L. (2005) Prospects for personalized cardiovascular medicine: the impact of genomics J Am Coll Cardiol 46, 1615–27. 8. Zhang, W., Huang, S., and Dolan, E. (2008) Integrating epigenomics into

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Chapter 13 Diagnosis and Management of Sickle Cell Disorders Shaker A. Mousa and Mohamad H. Qari Abstract Sickle cell disease (SCD) is a wide-spread inherited hemolytic anemia that is due to a point mutation leading to a valine/glutamic acid substitution in the β-globin chain, causing a spectrum of clinical manifestations in addition to hemolysis and anemia. Acute painful crisis is a common sequela that can cause significant morbidity and negatively impact the patient’s quality of life. Remarkable improvements in our understanding of the pathogenesis of this clinical syndrome and the role of cell adhesion, inflammation, and coagulation in acute painful crisis have led to changes in the management of pain. Due to the endemic nature of SCD in various parts of the Middle East, a group of physicians and scientists from the United States and Middle East recently met to draw up a set of suggested guidelines for the management of acute painful crisis that are reflective of local and international experience. This chapter brings together a detailed etiology, pathophysiology, and clinical presentation of SCD, including the differential diagnoses of pain associated with the disease, with evidence-based recommendations for pain management and the potential impact of low-molecular weight heparin (LMWH), from the perspective of physicians and scientists with long-term experience in the management of a large number of SCD patients. Key words: Painful crisis, vaso-occlusive crisis, sickle cell, guidelines, low-molecular weight heparin, prophylaxis, diagnosis, management.

1. Introduction The inherited disorders of hemoglobin are the most common gene disorders, and it is estimated that 7% of the world’s population are carriers. Approximately 300,000 children worldwide are born with documented sickle cell disease (SCD) every year. Sickling disorders are found frequently in the Afro-Caribbean populations and sporadically throughout the Mediterranean region, India, and the Middle East (1). S.A. Mousa (ed.), Anticoagulants, Antiplatelets, and Thrombolytics, Methods in Molecular Biology 663, DOI 10.1007/978-1-60761-803-4_13, © Springer Science+Business Media, LLC 2003, 2010

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SCD is due to a genetic mutation that results in an amino acid substitution of valine for glutamic acid at position 6 of the adult β-globin chain. In the homozygous form, this substitution results in polymerization of hemoglobin upon deoxygenation, leading to deformed dense red blood cells in sickle cell anemia (SCA) patients. The predominant pathophysiological feature of homozygous SCA is vaso-occlusion, which leads to acute and chronic complications such as painful crises, increased risk of infection, acute chest syndrome, stroke, and severe pain episodes with dramatic hemolysis, which progress to involve multiple organs, including the central nervous system, cardiovascular system, lung, liver, bone, skin, and kidneys (2). The epidemiology of SCA in the Arab region and the high prevalence of the mutation are well documented. In Saudi Arabia, the overall prevalence of the sickle cell trait is 4.20%, with wide variation from region to region. In Oman, the prevalence of the sickle cell trait is 6%, and in Bahrain, 11.2% of the population are sickle cell gene carriers (3–5). Extensive studies conducted over several years in different provinces of Saudi Arabia have revealed a wide distribution pattern of the hemoglobin S (HbS) gene in different provinces. The overall prevalence of HbS carrier status in Saudi Arabia varies from 0–1% in the northern and central regions to approximately 7% in the western, 12% in the southern, and nearly 25% in some parts of the eastern region (6, 7). The gene frequency of HbS in the populations of the Gulf region and other middle eastern states is one of the highest in the world. This chapter will present a consensus opinion on the management of painful vaso-occlusive crisis based on the recommendations of a panel of physicians and scientists from these regions. The objective is to provide guidelines for the local practicing hematologist and non-hematologist who might not be exposed to large numbers of SCA patients, with the goal of standardizing preventive immediate care in the emergency room, upon admission, and upon follow-up. The serious morbidity and hampered quality of life inflicted by painful crisis creates an urgent need for well-informed guidelines for the management and treatment of patients suffering from SCD at the local level.

2. Pathogenesis The clinical hallmark of SCA is the painful acute “crisis,” which despite therapeutic advances, continues to be a treatment challenge. Such crises occur with variable frequency and duration, and they commonly require hospitalization (8).

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The pathobiology of SCD is characterized by episodic vascular occlusion, with multiple inciting events beyond actual HbS polymerization and mechanical obstruction induced by sickled red blood cells (RBCs). An important mechanism of pain induction is thought to involve bone marrow vasculature infarction, leading to the release of inflammatory mediators that in turn stimulate afferent nerve fibers and cause pain. Vaso-occlusion also involves adherence of circulating blood elements, such as leukocytes, to endothelial cells, hyper-coagulability, endothelial dysfunction, altered nitric oxide metabolism, and ischemia reperfusion injury (9–11). 2.1. Increased Red Cell Adhesion

Abnormal interactions between erythrocytes and the endothelium are a primary initiating factor in the development of microvascular occlusions in SCD. This view is supported by reports of a significant correlation between clinical severity of SCD and extent of RBC adhesiveness (9, 12).

2.2. Increased Leukocyte Adhesion

Neutrophils are likely to be an important factor in causing microvascular sickle cell trapping and consequent vaso-occlusion. Sickle cells appear to be more adherent to neutrophils than normal RBCs. Sickle cells also induce neutrophil oxidative activity, which might be important in neutrophil-induced tissue damage during vaso-occlusive episodes (13).

2.3. Hyper-Coagulability

Patients with SCD exhibit high plasma levels of markers of thrombin generation, depletion of natural anticoagulant proteins, reduced activity of the fibrinolytic system, and increased tissue factor expression, even in the non-crisis steady state. Platelets and other cellular elements are also chronically activated, which might predispose the patient to thromboembolic manifestations (14, 15). Because of the increased generation of thrombin and fibrin and the increased tissue factor pro-coagulant activity in patients, SCD is often described as a hyper-coagulable state (16, 17). Recently, it was shown that compared to healthy ageand race-matched controls, patients with SCD had higher levels of plasma markers of coagulation activation (D-dimers, F1+2, and thrombin-anti-thrombin III complex) (18).

2.4. Reperfusion Injury and Nitric Oxide

A central aspect of sickle cell vasculopathy is the impairment of endothelial regulation of vasomotor tone, thrombosis, and inflammation (9). Intermittent vascular occlusion in SCA patients leads to reperfusion injury, which is associated with granulocyte accumulation and the enhanced production of reactive oxygen species. The recruitment of nitric oxide (NO) to counteract the resultant oxidative stress reactions results in a reduction in NO bioavailability and contributes to vascular dysfunction in SCD (10).

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2.5. Role of Inflammatory Mediators

There is an emerging consensus that a pro-inflammatory state contributes to the vaso-occlusive complications associated with SCD. Tissue damage due to vaso-occlusion results in the release of numerous inflammatory mediators that initiate the transmission of painful stimuli and the perception of pain. Plasma cytokines and related factors represent a burgeoning area of inquiry related to the pathogenesis in SCD. Cytokines derived from platelets, white blood cells, and endothelial cells have been implicated in the development of several sequelae of the disease (19, 20). In as much as sickle cell adhesion to the endothelium plays a role in vaso-occlusion, the existence of such diverse mechanisms of adhesion presents an enormous challenge in terms of identifying physiologically relevant therapeutic targets. Interestingly, a recent study has suggested that targeting a specific adhesion pathway may be sufficient to reduce vasoocclusion (21). In summary, the vascular pathophysiology of SCD is due to the combination of many factors, including red and white blood cell adhesion to the endothelium, increased coagulation, and homeostatic perturbation. The vascular endothelium is central to disease pathogenesis because it presents adhesion molecules for the attachment of blood cells, balances the pro-coagulant and anticoagulant properties of the vessel wall, and regulates vascular homeostasis by synthesizing vaso-constricting and vaso-dilating substances. Intermittent vascular occlusion in SCD leads to reperfusion injury, which is associated with granulocyte accumulation and enhanced production of reactive oxygen species. The

Table 13.1 Rational for tinzaparin in SCDa Tinzaparin: • Exerts favorable pharmacodynamic effects as compared to other LMWHs on a variety of cellular factors, including endothelial TFPI, vWF, TNF-α, NO modulation, and P/L-selectin • Inhibits matrix degrading enzymes more effectively than other LMWHs • Reduces oxidative stress by inhibiting the generation of reactive oxygen species • Induces long-lasting increases in plasma TFPI and NO • Has favorable anticoagulant efficacy and safety profiles • Has a once daily pharmacokinetic profile • Does not require any special dose adjustment or monitoring in special patient populations, including severe renal failure patients a see Refs. (8, 45–55).

LMWH, low-molecular weight heparin; TFPI, tissue factor pathway inhibitor; vWF, vonWillebrand factor; TNF, tumor necrosis factor; NO, nitric oxide; SCD, sickle cell disease.

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recruitment of NO to counteract the resultant oxidative stress reactions results in reduced NO bioavailability and contributes to vascular dysfunction in SCD. Table 13.1 illustrates the impact of the low molecular weight heparin (LMWH) tinzaparin on many of these complex processes.

3. Clinical Presentation of the Painful Crisis 3.1. Factors Precipitating Painful Crisis

Pain can be precipitated by hypoxia, infection, fever, acidosis, dehydration, pregnancy, menstruation, obstructive sleep apnea, and exposure to cold or abrupt weather changes. Coinheritance of the methylenetetrahydrofolate reductase (MTHFR) C677T mutation also predisposes SCA patients to pain (22). Patients also cite anxiety, depression, alcohol consumption, and physical exhaustion. Pain can be precipitated by co-morbidities such as sarcoidosis, diabetes mellitus, cholecystitis, and herpes; in many instances, no precipitating event is identified (23–25).

3.2. Effects of Pain on Quality of Life and Survival

SCD is chronic and lifelong. Individuals are most often well, but their lives are punctuated by periodic painful attacks, and they are at increased risk of other medical complications and premature death (26). Several studies have examined the relationship between pain and quality of life, and current emphasis in treatment is on managing distress, pain relief, and psychological support (27). The average life expectancy of males and females with SCD is 42 and 48, respectively (26). Individuals who experience >6 episodes per year have a reduced survival rate compared to those who experience less frequent events. However, with good health care, many individuals with SCA maintain reasonably good health most of the time and live productive lives. In fact, in the past 30 years, the life expectancy of individuals with SCA has increased (23).

3.3. Characteristics of Pain

The acute painful crisis is characterized by a sudden onset of pain that may start in any part of the body. The pain is variable from mild to severe with excruciating deep pain that is felt in the bones and soft tissues. Acute painful crisis is distinct from other varieties of pain that can arise due to complications of sickle cell anemia, such as acute chest syndrome, priapism, splenic and hepatic sequestration, hand foot syndrome, arthritis, and abdominal pain caused by calcular cholecystitis. Before embarking on a

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diagnosis of acute painful crisis, it is important that these other causes of pain be ruled out (23).

4. Management of Painful Episodes

Figures 13.1 and 13.2 present summary guidelines for managing painful crisis in adults and children, respectively. These guidelines

Pain is severe and not responding to oral analgesia (pain assessment tools might include visual analog scale, verbal scale etc.)

Rapid clinical assessment, clinical laboratory chemistry, blood film, blood gases

Insert IV line/encourage oral fluid intake Morphine (0.1 mg/kg IV or SC every 20 minutes)

Pain controlled Maintenance: Morphine (0.05–0.1 mg/kg/2–4hrs SC/IV or PO; alternatively, use PCA) Adjuvant therapy: 1. Hydroxyurea 2. Tinzaparin (Inno hep®) (175 IU/kg daily x–27 days) 3. Ibuprofen, Paracetemol (PO or IV), Diclofenac, Ketorlac 4. Laxative: Lactose (10 ml 1.0 bid) 5. Antiemetic: Prochloroperazine (5–10 mg PO tid) or Metochlopramide 6. Anxiolytic: Haloperidol (1–3 mg PO or I/M bid in case of anxiety or agitation) 7. Fentanyl (25 mcg, patch will last for 3 days)

Pain persists

1. Diamorphin (0.01 mg/kg IV or SC); or Hydromorphine 2. Rule out other causes of severe pain 3. Involve pain management team and social team 4. Consider transfer to ICU 5. Exchange transfuse the patient and consider caudal analgesic if the patient has lower body pain (PCA should be the guidance for opioid administration to minimize adverse effects)

Monitor pain, vital signs and O2 saturation every 30 min; when pain is controlled, repeat monitoring every 2 hours

If respiratory rate <10/min or O2 saturation <90% or patient sedated, give Naloxone (0.1 mg repeated every 2 min as necessary) and stop sedatives

Pain improved based on standardized pain assessment tools; discharge patient and arrange home care plan with oral analgesics

Fig. 13.1. Management of adults with painful crisis. IV, intravenous; SC, subcutaneous; PO, per oral; PCA, patientcontrolled analgesia; bid, twice daily; tid, thrice daily; IM, intramuscular.

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Rapid clinical assessment Tests (Table 2) Insert IV line/encourage oral fluid intake

Mild to moderate pain: Paracetamol (15 mg/Kg/dose) + Codeine (1 mg/kg/dose PO q4 hours) ± Ibuprofen (5–10 mg/kg/dose PO q6–8 hours; max 2400 mg/day)

Improvement

Pain Persists

Moderate to severe pain: Morphine (0.1–0.15 mg/kg/dose, repeat hourly) Adjuvant therapy: 1. Hydroxyurae 2. Tinzaparin (200–240 IU/kg SC once daily) 3. Ibuprofen, Paracetemol (PO or IV), Diclofenac, Ketorlac 4. Laxative: Lactulose (10 ml 1.0 bid) 5. Antiemetic: Prochloroperazine (5–10 mg PO tid) 6. Anxiolytic: Haloperidol (1–3 mg PO or I/M bid)

Maintenance: additional 0.05 mg/kg Morphine q1–2 hours until improvement.

Change to oral analgesia, Paracetamol or Ibuprofen

Discharge and arrange home care plan

Fig. 13.2. Management of painful crisis in children (less than 11 years of age). IV, intravenous; SC, subcutaneous; PO, per oral; bid, twice daily; tid, thrice daily; IM, intramuscular.

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have been drawn up based on the recommendations and experience of a panel of international basic and clinical scientists and local practitioners from several major hospitals in Saudi Arabia and the Gulf region, as well as a number of excellent reviews detailing the clinical management of painful crisis in SCA patients (11, 24, 28–43). 4.1. Management at Presentation

Patients should be seen immediately by a physician and a thorough history and examination should be carried out. Routine laboratory testing might not be necessary in patients with uncomplicated vaso-occlusive crisis. If a patient has symptoms that are severe enough to warrant hospitalization, laboratory tests should include a complete blood count, reticulocyte count, and urinalysis. If fever is present, a chest radiograph should be obtained, and urine, sputum, and blood should be cultured to rule out infection. Fever is common in patients with uncomplicated vasoocclusive crisis and does not necessarily indicate the presence of an underlying infection (33). If the patient experiences severe abdominal pain, recurrent vomiting, respiratory symptoms, neurological signs of paresis or paralysis, acute joint swelling, priapism, or an abrupt fall in hemoglobin, the treating physician should be alerted, and the etiology should be identified and treated (23). Comprehensive approaches to ambulatory and inpatient pain management have been addressed in a number of publications (20, 29, 33). These reports are a particularly good resource for establishing clinical pathways for hospital-based pain management in patients with SCD. Recommendations include the timely use of opioid medications for moderate to severe pain and avoidance of meperidine (Demerol) (11, 30–33, 37).

4.2. Home-Based Versus Hospital Treatment

The best approach to managing pain is aggressive analgesic therapy with frequent reassessment of effectiveness using a standardized pain assessment tool. The treatment of milder episodes of pain can be achieved at home with oral fluids, oral analgesics such as paracetamol, non-steroidal anti-inflammatory drugs (NSAIDs) (ibuprofen in particular for children) (37), and comfort measures, such as heating pads. For the patient who has persistent pain, codeine or tramadol is recommended. When homebased management fails to adequately alleviate pain, it is essential that patients undergo rapid triage, physical assessment, and aggressive, appropriately monitored analgesia (31).

4.3. Hospitalization

For severe pain, the patient will require hospitalization. Hydration and parenteral opioids such as morphine are indicated and usually administered by scheduled around-the-clock dosing or patientcontrolled analgesia (PCA) (29, 31, 33). During admission and episodes of severe pain, life-threatening complications may develop rapidly and often are heralded by relatively sudden clinical

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Table 13.2 Routine clinical investigation at presentation • Complete blood count and differential • Reticulocyte count • Blood chemistry profile • Urine analysis and cultures • Chest X-ray • Pulse oxymetry • In pregnant patients the following tests are also required: ◦ Hemoglobin electrophoresis ◦ Screening for viral hepatitis ◦ Vaginal swabs and endocervical smears ◦ Blood grouping and antibody screening ◦ Iron studies (TIBC, ferritin) ◦ Folate and vitamin 12 levels ◦ Thyroid function test

changes, such as increased oxygen requirement, altered mental status, or decreased hemoglobin levels or platelet counts (39, 41). Guidelines for routine clinical measures to be taken at presentation are summarized in Table 13.2. Opioids should not be withheld because of the unfounded fear of addiction. A recent retrospective evaluation of pain assessment and treatment for acute vaso-occlusive episodes in children with SCD concluded that despite opiate dosing within recommended guidelines, mean pain scores remained in the moderate to severe range for several days following hospitalization for vasoocclusive episodes (35). The fact that pain is severe and protracted and involves complex underlying etiologies, including impaired cell–cell interactions, increased production of pro-inflammatory mediators, hemostatic imbalance, and hyper-coagulability, paves the way for the development of alternative approaches to therapy, including the use of anti-adhesion, anti-inflammatory, and anticoagulation agents. Recently, the LMWH tinzaparin, given as a supplement to opioid treatment, was found to significantly shorten the protracted course of pain in a randomized, controlled, double-blind study (8). Thus, tinzaparin is justified for use in the treatment of acute painful crisis in SCA. The clinical effects of tinzaparin are most likely due to its favorable pharmacodynamic effects on a variety of cellular factors, including endothelial tissue factor pathway inhibitor (TFPI), tumor necrosis factor (TNF)-α, NO activity, von Willebrand factor (vWF), matrix degrading enzymes, and P-selectin (44–53). Long-lasting increases in

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plasma concentrations of TFPI and NO levels induced by tinzaparin in particular would contribute favorable anticoagulant effects. Given once daily, tinzaparin does not require any special monitoring (8, 54, 55). However, further trials are required before making a general recommendation. 4.4. Potential Prophylaxic and Therapeutic Impact of Anti-thrombotic Agents on SCD

Aspirin, standard heparin, and warfarin have been used for the treatment of acute painful crisis without conclusive results (44, 46). Tinzaparin has been show to have anti-thrombotic, antiinflammatory, and vascular protective effects in obese and healthy subjects and is justified in the treatment of acute painful crisis in SCA based on a randomized, double-blind clinical trial (8, 48, 51–53) (see Table 13.1).

4.5. Ancillary Measures

Ancillary measures to reduce the incidence of complications during acute painful crisis include the use of frequent incentive spirometry while the patient is awake to encourage deeper inspiratory effort, and avoidance of fluid overload by limiting overall intake to 1.0–1.5 times the maintenance need (56). Oxygen supplementation is not needed unless hypoxemia is present. Close monitoring of oxygen saturation and respiratory status, with particular attention to excessive sedation, is necessary (57). Other adjuvants include antihistamines, anticoagulants, antidepressants, benzodiazepines, and anticonvulsants. This is a heterogeneous group of compounds that potentiate the analgesic and ameliorate the side effects of opioids, and exert mild analgesic effects. The role of selective serotonin reuptake inhibitors in SCA is not clear at present. Adjuvants must be used with care, and patients should be monitored carefully when receiving them. Adjuvant therapy can also have adverse effects, some of which precipitate or worsen the manifestations of SCA. Other considerations include maintaining adequate (but not excessive) hydration, the provision of heating pads, massages, warm baths, and other comfort measures, monitoring of oxygenation and cardiopulmonary status, and close observation of other potential complications, particularly acute chest syndrome (56–63).

4.6. Discharge Criteria

The patient is considered ready for discharge when he/she can tolerate oral fluids and medications, pain is controlled by PO medication, and concurrent problems are resolved. Long hospital stays are often complicated by hospital-acquired infections.

4.7. Rehabilitation and Psychological Considerations in the Management of SCD

Recurrent pain has an immeasurably negative impact on daily activities, school and work performance, social interactions and relationships, mood, quality of life, and recreation activities. The psychosocial aspects of chronic pain are complex and often are

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Table 13.3 Recommended physical and psychosocial services for SCA patients • Establishment of a dedicated unit for the care of SCD • Establishment of a dedicated multi-disciplinary team of specialized doctors, nurses, physical therapist, psychologist/psychiatrist, social workers, pain management specialist • Good spectrum of pain medication drugs • Physiotherapy • TENS therapy • Behavioral/cognitive therapy • Participation in national societies/support groups • Acupuncture/acupressure may be beneficial • Distraction and entertainment, including videos and other media • Improved clinical outcomes in the management of SCD crisis • Medical and health education of medical staff and patient on: ◦ Forms of and preventing exposure to hypoxia (i.e., altitude, smoking) ◦ Habits and the use of charcoal in heating within closed areas ◦ Avoidance of excessive physical stress and competitive exercise ◦ Avoidance of sudden changes in temperature ◦ Avoidance of exposure to infection ◦ Avoidance of dehydration SCA, sickle cell anemia; TENS, transcutaneous electrical nerve stimulation

not appropriately addressed. A patient with chronic and recurrent pain must undergo a thorough psychosocial assessment to help define stressors and co-morbidities (i.e., depression), coping strategies, and support systems (64). Many patients with SCD suffer from physical and psychological stressors/complications due to the disease or disease therapy. These complications can include divorce, loss of family ties and friendship, unemployment due to interruption of work, dependence on social welfare support, and stereotyping of patient problems with drug-seeking behavior. Generally, there is no consistent plan for disease management and follow-up by physicians who are not experienced in the treatment of SCD. This lack of a consistent plan, in addition to the disabling complications of SCD such as chronic pain syndrome, avascular necrosis, arthropathies, and other skeletal manifestations, can result in depression and low self-esteem. Table 13.3 presents an example of a well-coordinated structured program based on the physical and psychological aspects of SCD (64–67).

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5. Pregnancy and Sickle Cell Anemia

Pregnancy increases the complications of SCA for the mother, fetus, and neonate, but there have been significant improvements in outcomes (68–70). The corner stone of management during pregnancy involves the hematologist and the obstetrician, as the mother needs regular monthly follow-ups and close fetal monitoring. All pregnant SCA patients should receive folic acid from the beginning of pregnancy. Immunization history should be current, particularly hepatitis B, pneumococcal, and influenza vaccinations. Asymptomatic bacteruria must be treated as well as any other active infection. Patient must be instructed to adequately hydrate and avoid physical and psychological stress.

5.1. Management of Sickle Cell Patients During Pregnancy

Forty-eight percent of women with SCD experience a crisis during pregnancy (69, 71). Painful sickle cell crisis requires admission and treatment with intravenous hydration and pain control. Analgesia should be adjusted based on the patient’s response, with rapid relief being the goal. NSAIDs are not the therapy of choice in pregnant patients who have a mild crisis because of the concern for premature closure of the ductus arteriosus at advanced gestational age. Rather, opiates, preferably morphine, are recommended. Patients should be on a scheduled dosage with additional boluses if needed. Oxygen therapy should be given if oxygen saturation is less than the patient’s known steady state. Adjuvant therapy such as stool softeners, antipruritics, and anxiolytics/sedatives should be considered (72). Blood transfusion may be indicated if signs or symptoms of anemia are present (tachycardia, tachypnea, dyspnea, fatigue, decreasing hemoglobin, a low reticulocyte count of <100×109/L). Blood should be leukopoor and antigen matched. In extreme cases, exchange transfusions may be required (73).

5.2. LMWH in Pregnancy and Sickle Cell Anemia

LMWHs are regarded as attractive alternatives to unfractionated heparin (UFH) as anticoagulants during pregnancy due to their clinical advantages and their association with a lower incidence of bleeding, osteoporosis, and heparin-induced thrombocytopenia (HIT). Many published reviews confirm that LMWHs are a safe alternative to UFH as anticoagulants during pregnancy (74–79). R Tinzaparin sodium, the active ingredient of innohep , has been used extensively in pregnant women at risk of developing or with a history of venous thromboembolism (VTE) and has proven to be a safe and effective pharmacologic agent. A recent multicentre, prospective, dose-finding clinical study report published in the American Journal of Obstetrics and Gynaecology

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R documented the safety and efficacy of innohep treatment of high-risk pregnancy cases of various aetiologies by also measuring anti-Xa levels throughout the gestation period and beyond (80). A treatment dose of 175 IU/kg/day and a prophylactic dose of 75 IU/kg/day reliably achieved target activities that were sufficient and safe, and there were stable gestational tinzaparin requirements (no dose adjustments). In addition, there were no apparent clinical effects of treatment on bone density. The use of tinzaparin during crises in SCA patients has been documented (8); however, the issue of its use during crises in pregnant SCA patients has not been thoroughly investigated. Recent guidelines acknowledge that SCA pregnant patients are at risk of developing VTE and, accordingly, a prophylactic dose of LMWH can be given throughout the pregnancy and postpartum (81).

5.3. Management During Labor

Generally, delivery can be accomplished vaginally, with cesarean section reserved for obstetric indications. Spontaneous labor is preferable because sickle cell crisis has been reported to be associated with induction of labor. There is a concern that the use of prostaglandins may lead to cell sickling, and therefore, should be used with caution (73).

5.4. Blood Transfusion

Prophylactic blood transfusions have been used to treat pregnant women who have sickle disorders. Currently, their use is controversial. A comparative trial of prophylactic versus on demandbased transfusion showed no difference in prenatal outcome between the two groups. The group that was transfused had a lower incidence of painful crisis; however, other medical and obstetric complications occurred with equal frequency (73).

6. Conclusions SCD, with its burden of excruciating pain associated with sickle cell crisis, is the most common globin gene disorder and a major health problem. The prediction in very early childhood of later severe disease could justify the early use of disease-modifying procedures or interventions, such as hydroxyurea treatment, chronic transfusions, or stem cell transplantation. The hazards of these treatments vary greatly, particularly as compared to preventive and supportive management alone. Accurate and reliable early predictors would provide the opportunity to better balance the risks of these interventions with risks of the disease itself and might reduce the frequency of hospitalization and blood transfusion, the incidence of pain, and the occurrence of acute chest

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syndrome and pulmonary hypertension in patients with SCD. A randomized, double-blind clinical study of the LMWH tinzaparin demonstrated significant and rapid pain resolution, underscoring the urgent need for additional therapeutic strategies designed to counteract the endothelial dysfunction and the inflammatory, hypercoagulation, and oxidative abnormalities of SCD. Currently, there is an unmet medical need for better management of SCD patients. Not covered by this review, but of special consideration in the management of SCD patients, are renal failure, addiction to opiates, and peri-operative painful crisis management. References 1. Jeremiah, Z.A. (2006) Abnormal haemoglobin variants, ABO and Rh blood groups among student of African descent in Port Harcourt, Nigeria Afr Health Sci 6, 177–81. 2. Hiran, S. (2005) Multiorgan dysfunction syndrome in sickle cell disease J Assoc Physic India 53, 19–22. 3. Alhamdan, N.A., Almazrou, Y.Y., Alswaidi, F.M., and Choudhry, A.J. (2007) Premarital screening for thalassemia and sickle cell disease in Saudi Arabia Genet Med 9, 372–7. 4. Al-Riyami, A., and Ebrahim, G.J. (2003) Genetic blood disorders survey in the Sultanate of Oman J Trop Pediatr 49(Suppl 1), i1–20. 5. Mohammed, A.M., Al-Hilli, F., Nadkarni, K.V., Bhagwat, G.P., and Bapat, J.P. (1992) Hemoglobinopathies and glucose-6phosphate dehydrogenase deficiency in hospital births in Bahrain Ann Saudi Med 12, 536–9. 6. el-Hazmi, M.A., and Warsy, A.S. (1999) Appraisal of sickle-cell and thalassaemia genes in Saudi Arabia East Mediterr Health J 5, 1147–53. 7. El-Hazmi, M.A., and Warsy, A.S. (1994) The frequency of glucose-6-phosphate dehydrogenase phenotypes and sickle cell genes in Al-Qatif oasis Ann Saudi Med 14, 491–4. 8. Qari, M.H., Aljaouni, S.K., Alardawi, M.S., Fatani, H., Alsayes, F.M., Zografos, P., Alsaigh, M., Alalfi, A., Alamin, M., Gadi, A., and Mousa, S.A. (2007) Reduction of painful vaso-occlusive crisis of sickle cell anaemia by tinzaparin in a double-blind randomized trial Thromb Haemost 98, 392–6. 9. Aslan, M., and Freeman, B.A. (2007) Redoxdependent impairment of vascular function in sickle cell disease Free Radic Biol Med 43, 1469–83. 10. Hebbel, R.P., Osarogiagbon, R., and Kaul, D. (2004) The endothelial biology of

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SUBJECT INDEX

A

Animal venous thrombosis models chemically-induced thrombosis models . . . . . . . . . . . . 54 laser-induced thrombosis models . . . . . . . . . . . . . . . . . . 58 stasis-thrombosis model . . . . . . . . . . . . . . . . . . . . . . . 31, 58 vena-caval ligation model . . . . . . . . . . . . . . . . . . . . . . . . . 47 vessel-wall damage models . . . . . . . . . . . . . . . . . 31–32, 64 Anticoagulants Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50, 89 heparin-induced thrombocytopenia (HIT). . . . . . . .136, 140, 142–144, 146–147, 158, 171, 236, 302 latest developments . . . . . . . . . . . . . . . . . . . . . . 47, 51, 123, 142, 146, 235–236 tumor factors predicting sensitivity to . . . . . . . . . . . . . 111 Antifactor Xa agents . . . . . . . . . . . . . . . . . . . . . . . . . . 111–113, 163–164, 175, 194, 248, 303 Antigen assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136–138 Antiheparin-PF4 antibody . . . . . . . . . . . . 133–138, 140–141 enzyme-linked immunosorbent assays (ELISA) . . . . . . . . . . . . . . . . . . . . . . . 137–138, 148 Anti-integrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262 potential drug discovery target . . . . . . . . . . . . . . . . . . . 262 Antiplatelet efficacy assays . . . . . . . . . . . . . . . . . . . . . . 269–271 Antiplatelets ADP receptor antagonists . . . . . . . . . . . . . . . . . . . 204, 216 bleeding risks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222, 226 natural . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 Anti-platelet therapy acute coronary syndrome (ACS) . . . . . . . . . . . . . 187, 204 combination . . . . . . . . . . . . . . . . . 125, 204, 207–215, 226 aspirin and clopidogrel . . . . . . . . . . . . . . 204, 208–215 GPIIb/IIIa antagonists . . . . . . . . . . . . . . . . . . . . . . 207 Anti-selectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262–264 Antithrombin III (ATIII) . . . . . . . . . . . . . 111, 115–116, 235 Antithrombotic agents Novel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33, 51, 81, 88 bleeding tendency . . . . . . . . . . . . . . . . . . . . . . . . . 88–89 thrombosis animal models . . . . . . . . . . . . . . . . . 88–89 discovery . . . . . . . . . . . . . . . . . . . . . . . . . 33, 89, 92, 204 Antithrombotics . . . . . . . . 1–26, 29–94, 142, 144, 147–148, 158–159, 162, 164, 166, 168, 170, 204, 216, 229–237 Antitissue factor . . . . . . . . . . . . . . . . . 110, 161–162, 251, 255 Anti-Xa agents . . . . 111–113, 163–164, 175, 194, 248, 303 APC, see Activated protein C (APC) Argatroban (Novastan) acute venous thromboembolism . . . . . . . . . . . . . . . 88, 144 antithrombotic efficacy . . . . . . . . . . . . . . . . . . . . . . . . 88, 92 co-therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 heparin-induced thrombocytopenia (HIT) . . . 143–144, 158, 172 platelet aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . 12, 149 Thrombosis . . . . . . . . . . . . . . . . . . . . . . . . . 88, 91, 143–144

Abciximab clinical utility. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .268 heparin-induced thrombocytopenia (HIT) . . . . 207–208 metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254 percutaneous coronary intervention . . . . . . . . . . . . . . . 771 Activated factor VIIa, see Tissue factor (TF)/activated factor VIIa Activated partial thromboplastin time (aPTT) . . . . . . . . 2–3, 5, 51, 66, 69, 81–83, 88, 142–143, 147, 165–166, 168–170, 174, 236, 242, 248, 286 Activated protein C (APC) . . . . . . . . . . . . . . . . . 44, 163, 286 Acute myocardial infarction (AMI) animal models of . . . . . . . . . . . . . . . . . . . . . . . . . . 33–34, 91 low molecular weight heparin (LMWH) . . . . . . . . . . . 33 reperfusion therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Acute pulmonary embolism . . . . . . . . . . . 115, 158, 182, 188 Tinzaparin . . . . . . . . . . . . . . . . . . . . . . . 299–300, 302–303 Adverse drug reactions . . . . . . . . . . . . . . . . . . . . 278, 281, 287 Pharmacogenomics . . . . . . . . . . . . . . . . . . . . 278–279, 287 Alanine aminotransferase . . . . . . . . . . . . . . . . . . . . . . . 173, 184 Elevated . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173, 184 Alpha 2 integrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Alpha 4 integrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265–267 Alpha 4 beta 1 (VLA-1) . . . . . . . . . . . . . . . 264, 266–267 Alpha 4 beta . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7, 265–266 Alpha 5 beta 1 integrin . . . . . . . . . . . . . . . . . . . . . . . . . 267, 271 Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 bacterial infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 Alpha 5 beta 3 integrin . . . . . . . . . . . . . . . . . . . . 262, 268–270 Alpha 5 beta 5 mediated adhesion assay . . . . . . . . . . 270–271 Alpha 5 beta l–receptor–biotinylated fibronectin–binding assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 AMI, see Acute myocardial infarction (AMI) 3-aminopropyltriethoxysilane treated glass slides . . . . . . . 23 platelet immobilization on . . . . . . . . . . . . . . . . . . . . . . . . 23 preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Angiogenesis alpha 5 beta 1 integrin . . . . . . . . . . . . . . . . . . . . . . . . . . 267 coagulation . . . . . . . . . 111, 114–115, 119–120, 122–125, 127, 161–162, 232, 243, 248 low molecular weight heparin (LMWH) . . . . . 110–111 tissue factor (TF) . . . . . . . . . . . . . . . . . . . . . . . . . . 110–111, 115, 122, 243 tissue factor (TF)/activated factor VIIa . . . . . . . . . . . . 110 tumor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120, 123, 125, 243 Animal thrombosis models clinical correlates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 clinical relevance . . . . . . . . . . . . . . . . . . . . . . . . . . 30, 33, 88 selection of . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32–33, 89

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310 Subject Index

Arterial thrombosis . . . . . . . . . . . . . 44, 46–48, 53, 56–57, 82, 161–163, 165, 169, 181, 203, 222, 230, 236 copper coil-induced canine . . . . . . . . . . . . . . . . . . . . . . . 62 Arterial and venous thrombosis model (Harbauer) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45–48 Arteriovenous shunt thrombosis model . . . . . . . . . . . . . . . . 64 Aspirin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8, 12, 36, 38, 40, 44, 47, 51, 59, 77, 79, 88, 127, 147, 167, 184, 187, 194, 196, 203–215, 222–223, 230, 235, 254, 268–269, 300 heparin-induced thrombocytopenia (HIT) . . . . . . . . 147 Atherosclerotic plaques . . . . . . . . . . . . . . . . . . . . . . . . . . 48, 213 adhesion molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 ATIII, see Antithrombin III

B Bacterial infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 alpha 4 beta 1 integrin (VLA-1) . . . . . . . . . . . . . . . . . 266 alpha 5 beta 1 integrin . . . . . . . . . . . . . . . . . . . . . . . . . . 267 Bernard-Soulier syndrome (BSS) . . . . . . . . . . . . . . . . . . . . . 24 Beta 1 integrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266–267 alpha 4 beta 1 (VLA-1) . . . . . . . . . . . . . . . . . . . . . . . . . 266 alpha 5 beta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1, 266 bacterial infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 gastrointestinal disease . . . . . . . . . . . . . . . . . . . . . . . . . . 267 Beta 2 integrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 Beta 3 integrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268–269 Alpha IIb beta 3 (GPIIb/IIIa) . . . . . . . . . . . . . . . 268–269 antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268–269 clinical utility . . . . . . . . . . . . . . . . . . . . . . . . . . . 268–269 Alpha vs. beta . . . . . . . . . . . . . . . . . . . . . . . . . . . 3, 268–269 Bivalirudin (Hirulog) . . . . . . . . . . . . . . . . . . . . . . 142, 145, 172 Bleeding . . . . . . . . . . . . . . 30–32, 43, 46, 51, 57, 76–89, 120, 143–145, 147, 158, 162–170, 173–174, 183–184, 186–198, 205–207, 209–215, 222, 224–226, 230, 234, 236, 242, 250–251, 257, 268–269, 282–284, 286, 302 animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76–79 difficulties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88, 282 BSs . . . . . . . . . . . . . . . . . . . . . . . see Bernard-Soulier syndrome

C CAM, see Cell adhesion molecules (CAMs) Cancer Angiogenesis . . . . . . . . . . . 111, 120, 122–127, 162, 232, 243, 248, 262, 269 anti-platelet inhibitors . . . . . . . . . . . . . . . . . 244, 254, 256 coagulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122–124 tissue factor (TF) . . . . . . . . . . . . . . . . . . . . . . . 123–124 complications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125, 236 deep vein thrombosis (DVT) . . . . . . . . . . . . . . . . . 136 experimental models . . . . . . . . . . . . . . 111, 122, 162, 243 heparin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110, 118 low molecular weight heparin (LMWH) . . . . . . . . . . 110 metastasis, lung . . . . . . . . . . . . . . . . . . . . . . . 122, 162, 244 non-anticoagulant low molecular weight heparin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247–248 platelet-cancer cell adhesion . . . . . . . . . . . . . . . . . . . . . 120 thrombosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110, 118–125 tissue factor . . . . . . . . . . . . . 110, 121–122, 158, 242–243 tissue factor pathway inhibitor (TFPI) . . . . . . . . . . . . . . . . . . . . . . . . 110–111, 122 venous thromboembolism (VTE) . . . . . . . . . . . . 120–122 Canine arterial thrombosis . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 copper coil-induced . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Canine coronary artery thrombosis model . . . . . . . . . . 55–57 Canine injury-induced (electrolytic) arterial thrombosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 CAPRIE trial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 Cardiac surgery . . . . . . . . . . . . . 134–135, 142, 145, 147, 222 Lepirudin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142, 145, 147 Cardiopulmonary bypass (CPB) surgery . . . . . . . . . . . . . . . 71 Lepirudin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Cardiopulmonary bypass models . . . . . . . . . . . . . . . . . . 70–71 Cardiovascular disease (CVD) . . . . . . . . . 194–195, 204–205, 210–211, 215, 262, 272 pharmacogenomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 Catheter-induced thrombosis models . . . . . . . . . . . . . 64, 120 Cell adhesion molecules (CAMs) . . . . . . . 85, 111, 271–272, 280, 261, 265 as surrogate markers . . . . . . . . . . . . . . . . . . . 114, 262, 272 Cellular signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 tissue factor (TF)/activated factor VIIa . . . . . . . . . . . 124, 162, 243 Cerebral blood flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 Certoparin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 chemical characteristics. . . . . . . . . . . . . . . . . . . . . . . . . .111 CFR, see Cyclic flow reductions (CFR) Chandler loop . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5–6 Chemically-induced thrombosis models . . . . . . . . . . . . . . . 54 Chemotaxis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 heparin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Clopidogrel ACTIVE W study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184 acute myocardial infarction (acute MI) . . . . . . . . . . . . . 34 aspirin . . . . . . . . . . . . . . . . . . . . . . . . . . . 204–206, 208–215 asymptomatic patients . . . . . . . . . . . . . . . . . . . . . . 208, 214 cerebrovascular disease . . . . . . . . . . . . . . . . . 208, 212, 214 clinical trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208, 216 combination therapy . . . . . . . . . . 204–206, 208–213, 215 coronary artery bypass graft (CABG) surgery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208, 222 coronary artery disease . . . . . . . . . . . . . . . . . . . . . . . . . . 214 limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208, 222 non ST segment elevation . . . . . . . . . . . . . . . . . . . 212, 215 percutaneous coronary intervention (PCI) . . . . . . . . . . . . . . . . . . . . . . . . . . 208, 212, 224 stent thrombosis . . . . . . . . . . . . . . . . . . . . . . . 209, 224–225 Clotting Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242–243 Cancer (tumor) cell-induced . . . . . . . . . . . . 244–245, 247 indicators of . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244–245 Dabigatran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145, 172 defects in . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 mouse genetic models . . . . . . . . . . . . . . . . . . . . . 82, 85 Factor Xa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184–185 heparin-induced thrombocytopenia (HIT) . . . . . . . . 236 pharmacogenomics . . . . . . . . . . . . . . . . . . . . . . . . . 277–287 TGN–167 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173–174 thrombelastography (TEG) . . . . . . . . . . . . . . . . . . . . . . 3–5 thrombin induced clot formation . . . . . . . . . . . . . . . 55–57 canine coronary artery . . . . . . . . . . . . . . . . . . . . . 55–57 rabbit femoral artery . . . . . . . . . . . . . . . . . . . . . . 55–57 tissue factor (TF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 in vitro assays for formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 lysis . . . . . . . . . . . . . . . . . . . . . . . . . . 4–6, 13–14, 64, 83 strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Coagulation angiogenesis . . . . . . . . . . . . 111, 114, 120, 122–125, 127, 161–162, 243, 248

ANTICOAGULANTS, ANTIPLATELETS, AND THROMBOLYTICS Subject Index 311 animal models of . . . . . . . . . 30–33, 76, 89–90, 111, 120, 162–163, 166, 185, 241 cancer . . . . . . . . . 118–119, 121, 122–124, 127, 159, 231, 234, 236, 241–243, 248, 255 molecular markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242 in vitro models of . . . . . . . . . . . . . . . . . . . . 2, 5, 15, 30–33, 86, 90, 162, 164, 235 Coagulation disorders . . . . . . . . . . . . . . 31–32, 110, 114, 168, 262, 268, 279 mouse genetic models of . . . . . . . . . . . . . . . . . . . . 162, 247 Coagulation factor II (prothrombin) . . . . . . . . 281, 283, 285 Polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281, 285 Coagulation factor IX propeptide . . . . . . . . . . . . . . . . . . . . 286 mutation at ALA-l0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 Coagulation factor V Leiden. . . . . . . . . . . . . . . . . . . .285–286 R506Q . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285–286 Collagen . . . . . . . . . . . . 1, 6, 9–10, 17, 20–22, 25, 31, 37, 64, 66, 71–76, 84–86, 111, 203, 215, 222, 231–234, 280 Collagen-coated surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21 Copper coil-induced canine arterial thrombosis . . . . . . . . 62 Diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Coronary artery plaque . . . . . . . . . . . . . . . . . . . . . 48, 213, 272 platelet aggregation and monitoring . . . . . . . 6, 8, 75–76 transition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Coronary artery thrombosis model . . . . . . . . . . . . . . . . 49, 57 Canine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44, 57 Coronary blood flow . . . . . . . . . . . . . . . . . . . . . . . . . . 34–35, 41 Coronary thrombosis model (Folts Model) . . . . . . 33–45, 47 mechanical-induced . . . . . . . . . . . . . . . . . . . . . . . . . . 33–45 stenosis-induced . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Crohn’s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116, 267 CURE trial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208, 211–212, 214 Cyclic flow reductions (CFR) . . . . . . . . . . . . . . . . . . 34, 38, 91

D Deep venous thrombosis (DVT) . . . . . . . . 91, 110, 115, 119, 121, 127, 141, 158, 160–161, 163–165, 167–169, 171, 173, 182, 188–194, 196, 285–286 rivaroxaban . . . . . . . . . . . . . . . . . . . . . . . . . . . 160, 167–168, 188–194, 196 Dextran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54, 60, 79 fluorescein-labeled . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Diabetes Clopidogrel. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .208, 212 Prasugrel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208, 225 Warfarin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 DIC, see Disseminated intravascular coagulation (DIC) Direct thrombin inhibitors . . . . . . . . . . . . . . 54, 71, 142–145, 158, 170–172, 236 heparin-induced thrombocytopenia (HIT) . . . . . . . . . . . 142–145, 158, 171–172, 236 Disseminated intravascular coagulation (DIC) . . . . . . . . . . . . . . . . . . 69–70, 148, 161, 242 animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 Drug metabolizing enzymes. . . . . . . . . . . . . . . .281–282, 287 gene polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282 phase I enzymes . . . . . . . . . . . . . . . . . . . . . . . . 281–282 phase II enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282 Drug transporter gene polymorphisms . . . . . . . . . . . 282–283 DVT, see Deep venous thrombosis (DVT) DX-9065a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90, 165

E EAE, see Experimental autoimmune encephalomyelitis (EAE) Echistatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232–233 ECM, see Extracellular matrix Elderly . . . . . . . . . . . . 143, 165–166, 183, 186, 189, 196–197 ELISA, see Enzyme-linked immunosorbent assay (ELISA) Embryonic lethal phenotype factor II (prothrombin) . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 factor X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 thrombomodulin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .83 Endothelial nitric oxide synthase (eNOS) . . . . . . . . . . . . 285 Enoxaparin anticoagulant effects . . . . . . . . . . . . . . . . . . . 113, 164–165, 167–168, 187, 193, 247 coronary thrombosis models . . . . . . . . . . . . . . . . . . . . . . 44 heparin-induced thrombocytopenia (HIT) . . . . . . . . 148 hip replacement . . . . . . . . . . . 91, 168, 170, 173, 190–191 metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247–249 tumor cell-induced clotting . . . . . . . . . . . . . . . . . . . . . . 247 compared to . . . . . . . . . . . . 113, 166, 168–169, 173, 187, 189, 191, 193, 247 dabigatran . . . . . . . . . . . . . . . . . . . . . . . . . 167, 172–173 LY-517717 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 razaxaban . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 YM–150 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 Enzyme-linked immunosorbent assay (ELISA) . . . . . . . . . 136–138, 140–141, 149, 270 antiheparin-PF4 antibody-linked . . . . . . . 140–141, 148 EPIC trial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 EPILOG trial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 Eptifibatide (Integrilin) . . . . . . . . . . . . . . . . . 37, 91, 207, 268 Erythrocytes . . . . . . . . . . . 14–17, 20, 24–25, 43, 58, 66, 293 Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15–18, 22 Aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Deformability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Experimental autoimmune encephalomyelitis (EAE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266–267 Experimental thrombocytopenia or leucocytopenia . . . . . . . . . . . . . . . . . . . . 72–73, 254 Extracellular matrix (ECM) . . . . . . . . . 1, 21, 120, 261, 264, 266–267 Extracorporeal thrombosis models . . . . . . . . . . . . . . . . . 71–72

F Factor V Leiden, see Coagulation factor V Leiden Factor Xa catalytic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 coagulation propagation . . . . . . . . . . . . . . . . . . . . 163–170 molecular target . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 Factor Xa inhibitors clinical trials . . . . . . . . . . . . . . . . . . . . . . . . 89, 92, 146, 175 effect on coagulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 mechanism of action . . . . . . . . . . . . . . . . . . . 117, 233, 235 direct . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117, 235 indirect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 natural . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159, 161, 251 oral . . . . . . . . . . . . . . . . . . . . . 142, 150, 165, 167–168, 196 tissue factor pathway inhibitor (TFPI) . . . . . . . . . . . . 247 Factor XIII . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281, 285 Polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281, 285 Ferric chloride-induced carotid artery thrombosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53–55 Ferric/ferrous chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

ANTICOAGULANTS, ANTIPLATELETS, AND THROMBOLYTICS

312 Subject Index

Fibrinogen . . . . . . . . . 1–2, 5–6, 9–10, 12–13, 34, 37–38, 57, 68–69, 82–85, 87, 113, 115, 123, 143, 207, 230–236, 242–247, 264–265, 270, 280 adhesion assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270 Fibrinolytic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13, 61 euglobuliin lysis time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Fisetin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 FIX . . . . . . . . . . . . . . . . . . . . . . . . . . . 80–81, 87, 161, 170, 286 deletion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80–81 genetic model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 Flavanoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231–233 Flow cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . 17–19, 25, 139 Fluid shear stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2, 17, 24 assays of . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Fluorescein-labeled dextran, see Dextran Folts model, see Coronary thrombosis model Fondaparinux (fondaparinux sodium) drawbacks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164 heparin-induced thrombocytopenia (HIT) . . . 142, 146, 158, 164 indications and usage . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 venous thromboembolism (VTE) . . . . . . . 158, 163, 165 and warfarin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158, 164 compared to Idraparinux . . . . . . . . . . . 142, 146, 163–164 FVIIai . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

G Garlic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233–234 platelet aggregation . . . . . . . . . . . . . . . . . . . . . . . . . 233–234 Genes . . . . . . . . . . 73, 80–82, 84–85, 87, 183, 279, 281–286, 291–292, 303 in hematologic, hemostatic, and thrombotic disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . 278–279 Genistein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232 Genetic models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33, 79–88 thrombosis and hemostasis . . . . . . . . . . . . . . . . . . . . 79–88 Genetic polymorphisms . . . . . . . . . . 208, 237, 278, 281–282 drug effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 GPIIb/IIIA antagonists . . . . . . . . . . 5, 17, 22, 37, 44, 89–90, 207–208, 254, 268–269 oral . . . . . . . . . . . . . . . . . . . . . . 44, 207, 216, 254, 268–269 GUSTO V trial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210

H Harbauer model, see Arterial and venous thrombosis model (Harbauer) Hematological disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 Pharmacogenomics . . . . . . . . . . . . . . . . . . . . . . . . . 285–286 Hemophilia B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80–81 Hemostasis . . . . . . . . 32, 33, 73, 77–89, 114, 163, 169–170, 184, 221, 230, 237, 251 genetic models . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33, 77–88 Hemostatic disorders . . . . . . . . . . . . . . . . . . 32, 168, 208, 279 genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33, 279–280 Heparin anti-inflammatory . . . . . . . . . . . . 110, 115–118, 123, 126 asthma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111, 116 bioavailability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112–113 cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . 110–111, 118–127 deep vein thrombosis (DVT). . . . . . . . . . . . . . . . . . . . .110 endothelial release . . . . . . . . . . . . . . . . . . . . . . . . . . 120, 134 indications . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110–111, 113 inflammatory bowel disease . . . . . . . . . . . . . 111, 116–117

laser-induced thrombosis models . . . . . . . . . . . . . . . . . . 58 mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . 116–119, 122 metastasis . . . . . . . . . . . . . . . 110–111, 119–120, 122–127 oral . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119, 121, 123 pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89–90 platelet–cancer cell adhesion . . . . . . . . . . . . . . . . . . . . . 120 stasis thrombosis model . . . . . . . . . . . . . . . . . . . . . . . 31, 58 thrombosis . . . . . . . . . . . . . . . . . . . . . . . . 33, 110, 113–118 tissue factor pathway inhibitor (TFPI) . . . . 33, 110, 115 Heparin-induced thrombocytopenia (HIT) angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125–126 anticoagulants . . . . . . . . . . . 136, 140, 142–144, 146–147 antiheparin-PF4 antibody ELISA . . . . . . . . . . . 137, 141 clinical manifestations . . . . . . . . . . . . . . . . . . . . . . 135–136 flow cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 laboratory methods . . . . . . . . . . . . . . . . . . . . . . . . . 136–141 methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141–148 pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . 146–147 platelet aggregation assay . . . . . . . . . . . . . . . . . . . . . . . . . 90 serotonin release assay (SRA) . . . . . . . . . . . . . . . . 137–138 type I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136–138 type II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136–138 Heparin pentasaccharide . . . . . . . . . . . . . . . . . . . 116, 162–164 vs. low molecular weight heparin (LMWH) . . . . . . . . . . . . . . . . . 116–117, 134, 162 Heparin-platelet factor 4 (PF4) . . . . . . . 133–138, 140–141, 145–146, 148–149, 163–164, 280 Herbal supplements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 antiplatelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 drug interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . 230, 237 Hip replacement . . . . . . . . 145, 168, 170, 173, 181, 189–191 venous thromboembolism (VTE) . . . . . . . . . . . . 181, 189 Hirudin heparin-induced thrombocytopenia (HIT) . . . . . . . . 135 laser-induced thrombosis models . . . . . . . . . . . . . . . . . . 58 PEG-hirudin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44, 63 Hirulog, see Bivalirudin (Hirulog) HIT, see Heparin-induced thrombocytopenia (HIT) Human soluble selectin assays . . . . . . . . . . . . . . . . . . 263–264 Human soluble VCAM-I . . . . . . . . . . . . . . . . . . . . . . 272–273 Hypercoagulable assays . . . . . . . . . . . . . . . . . . . . . . . . . . 31, 147

I ICAM-I, see Inter-cellular adhesion molecule-1 (ICAM-1) Immobilized platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22–26 cell adhesion to . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22–26 Immunoglobulin . . . . . . . . . . . . . . . . 111, 136, 236, 261–262, 265–266, 271–273 Inflammation Heparin . . . . . . . . . . . . . . . . 110, 113–118, 122, 148–150, 159, 162, 295 low molecular weight heparin . . . . . . . . . . . . 44, 109–127 Thrombosis . . . . . . . . . . . 40, 85, 88, 110, 113–118, 123, 147–149, 158, 162, 293 Inflammatory bowel disease (IBD) . . . . . . . . . . . . . . . . . . 111, 116–117, 126, 267 Injury-induced (electrolytic) arterial thrombosis model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45–48 canine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 Innohep, see Tinzaparin (Innohep(tm)) Integrilin, see Eptifibatide (Integrilin) Integrin-based assays . . . . . . . . . . . . . . . . . . . . . . . . . . 269–271 Integrins alpha 2b beta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 alpha 4 integrins . . . . . . . . . . . . . . . . . . . . . . . . . . . 265–267

ANTICOAGULANTS, ANTIPLATELETS, AND THROMBOLYTICS Subject Index 313 angiogenesis . . . . . . . . . . . . . . . . . . . . . . 122, 126, 262, 267 bacterial infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 beta 1 integrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266–267 gastrointestinal disease . . . . . . . . . . . . . . . . . . . . . . . . . . 267 inflammatory disease . . . . . . . . . . . . . . . . . . . . . . . 266–267 integrin-based assays . . . . . . . . . . . . . . . . . . . . . . . 269–271 structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 vascular endothelial growth factor (VEGF) . . . 115, 127 vascular remodeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 Inter-cellular adhesion molecule-1 (ICAM-1) . . . . . . . . 114 Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Intravascular thrombosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . .72

K Knee replacement surgery fondaparinux sodium . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 prophylaxis . . . . . . . . . . . . . . . . . . . . . . . 146, 161, 166, 169 thromboembolic events . . . . . . . . . . . . . . . . . . . . . . . . . . 145 R ) . . . . . . . . . . . . . . . . . . . . . 296, 302 tinzaparin (lnnohep

L Large animal thrombosis models . . . . . . . . . . . . . . . . . . . . . . 29 Laser-induced thrombosis models . . . . . . . . . . . . . . . . . 57–59 Lepirudin acute venous thromboembolism (acute VTE) . . . . . . 158 heparin-induced thrombocytopenia (HIT). . . . . . . .142, 144–147, 149, 158, 172 Light transmittance aggregometry assay . . . . . . . . . 269–270 5-lipoxygenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86, 281 Low molecular weight heparin (LMWH) acute myocardial infarction (AMI) . . . . . . . . . . . . . . . 255 cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . .110–111, 118–127 chemical characteristics . . . . . . . . . . . . . . . . . . . . . 111, 113 combination strategies . . . . . . . . . . . . . . . . . . . . . . . . . . 136 complications of . . . . . . . . . . . . . . . . . . . . . . . . . . . 116, 125 deep vein thrombosis (DVT) . . . . . . . . 119, 121, 127 deep vein thrombosis (DVT) . . 110, 115, 119, 121, 127 heparin-induced thrombocytopenia (HIT) . . . . . . . . 158 inflammatory disease . . . . . . . . . . . . . . . . . . . 110, 114–116 metastasis . . . . . . . . . . . . . . . . . . . . . . . . 110–111, 119–120 pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187 thrombosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110, 113–118 tissue factor pathway inhibitor (TFPI) . . . . . . . 110, 122, 126–127 release . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112, 122, 127 venous thromboembolism (VTE) . . . . . . . . . . . . 120–121 vs. unfractionated heparin (UFH) . . . . . . . . . . . . . . . . . . . . 110, 112–113, 121

M MDR-1 gene polymorphisms . . . . . . . . . . . . . . . . . . . . . . . 283 Melagatran . . . . . . . . . . . . . . 44, 172–173, 175, 183–184, 188 see also Ximelagatran Metabolism . . . . . . . . . . . . . . 37, 90, 167, 182–183, 196–197, 226, 237, 282–287, 293 Pharmacogenomics . . . . . . . . . . . . . . . . . . . . . . . . . 283, 285 Metastasis blood-borne . . . . . . . . . . . . . . . . . . . . . . . . . . . 243–244, 252 coagulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241–254 experimental models . . . . . . . . . . . . . . . . . . . . . . . . 243, 254 heparin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .246–251 low molecular weight heparin (LMWH). . . . . . . . . . . . . . . . . . . . . .247–250, 255

Methylenetetrahydrofolate reductase gene (MTHFR) . . . . . . . . . . . . . . . . . . . . . . . . . 285, 295 C677T polymorphism . . . . . . . . . . . . . . . . . . . . . . 285, 295 Methylsulfonylmethane (MSM) . . . . . . . . . . . . . . . . . . . . . 235 Microvascular thrombosis trauma models . . . . . . . . . . . . . . 70 Monocytic THP-1 cell-platelet adhesion assay . . . . . . 22–24 Mothers Innohep(tm) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302 Labor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303 Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302 Multidrug-resistant (MDR-l) gene polymorphisms . . . . 283 Myocardial infarction acute, see Acute myocardial infarction (AMI) low molecular weight heparin (LMWH) . . . . . . . . . . . . . . . . . . . . . 110, 137, 142 tissue factor pathway inhibitor (TFPI) . . . . . . . . . . . . . . . . . . . . . . . . 110–113, 122 unfractionated heparin (UFH) . . . . . . . . . . . . . . . 110, 112

N NAPc2, see Nematode anticoagulant protein c2 (NAPc2) Nematode anticoagulant protein c2 (NAPc2) . . . . . . . . . 161, 175, 253 Novastan, see Argatroban (Novastan)

O Oral platelet GPIIb/III3a antagonists . . . . . . . . . . . . . . . . . . 5 Orbofiban . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23, 268

P Parallel-plate flow chamber . . . . . . . . . . . . . . . . . . . . . . . 20–24 PCI, see Percutaneous coronary intervention PECAM–1, see Platelet endothelial cell adhesion molecule-1 (PECAM-1) Percutaneous coronary intervention (PCI) . . . . . . . . 71, 143, 145, 206, 208–209, 211–212, 214–215, 224, 226 PE, see Pulmonary embolism (PE) PF4 . . . . . . . . . . . . . . . . . . . . . . . 133–138, 140–141, 145–146, 148–149, 163–164, 280 P-glycoprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282–283 Pharmacogenomics cardiovascular disease (CVD) . . . . . . . . . . . 277, 283, 285 definition of . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 hematological disorders . . . . . . . . . . . . . . . . . . . . . . . . . 285 single nucleotide polymorphisms (SNPs) . . . . . . . . . . 278 drug metabolizing enzymes . . . . . . . . . . . . . . . . . . 281 drug transporters . . . . . . . . . . . . . . . . . . . . . . . 282–283 warfarin therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . 282–284 Plaque . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48, 213, 272 coronary artery transition . . . . . . . . . . . . . . . . . 34, 49, 214 Platelet aggregation assays . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 laser-induced . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57–59 Born method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6–9 Platelet antagonists Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 endothelial cell migration . . . . . . . . . . . . . . . . . . . . . . . . 269 Platelet endothelial cell adhesion molecule-1 (PECAM-1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272 in vitro thrombosis flow models . . . . . . . . . . . . . . . . . . . 17 Platelet 125 1-fibrinogen-binding assay . . . . . . . . . . . . . . . 270 Platelet glycoprotein Ia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 gene polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286

ANTICOAGULANTS, ANTIPLATELETS, AND THROMBOLYTICS

314 Subject Index

Platelet glycoprotein IIIa . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 T393C gene polymorphism . . . . . . . . . . . . . . . . . . . . . . 286 Platelet GPIIb/IIIA antagonists . . . . . . . . . . . 5, 51, 89, 113, 207, 254, 268 chronic therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268–269 thromboembolic events . . . . . . . . . . . . . . . . . . . . . . . . . . 269 Platelet perfusion studies . . . . . . . . . . . . . . . . . . . . . . . . . 21–22 Platelet(s) Adhesion . . . . . . . . . . . . . . . . . . . 6, 19–25, 48, 58, 60, 79, 86, 203, 222, 232, 234 Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16–19 Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139, 147 Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 preparation platelet-poor plasma (PPP) . . . . . . . 4, 7–8, 248, 269 platelet-rich plasma (PRP) . . . . . . . . . . . . . . . . . . 6–9, 76, 137, 139, 239 washed platelets (WP) . . . . . . . . . . . . . . 6–9, 137, 139 Prasugrel acute coronary syndrome (ACS) . . . . . . . . . . . . . . . . . . 222 percutaneous coronary intervention (PCI) . . . . 224, 226 pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 renal impairment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226 Pregnancy Innohep(tm) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302–303 low molecular weight heparin (LMWH) . . . . . 302–303 sickle cell anemia . . . . . . . . . . . . . . . . . . . . . . . . . . . 302–303 venous thromboembolism (VTE) . . . . . . . . . . . . 302–303 Prothrombin, see Coagulation factor II (prothrombin) Pulmonary embolism (PE) Acute . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115, 158, 188 arterial blood gases (ABG) . . . . . . . . . . . . . . . . . . . . . . . . 43 heparin-induced thrombocytopenia (HIT) . . . . . . . . . . . . . . . . . . . . . . . . . 133–136, 158 Tinzaparin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 unfractionated heparin (UFH) . . . . . . . . . . . . . . . 121, 158

Q Quantitative sandwich immunoassay technique, see Enzyme-linked immunosorbent assay (ELISA)

R Rabbit femoral artery thrombosis . . . . . . . . . . . . . . . . . . . . . 91 Rat jugular-vein clamping thrombosis model . . . . . . . . . . . 31 Recombinant inactivated FVIIa (FVIIai) . . . . . . . . . . . . . 161 Recombinant nematode anticoagulant protein c2 (NAPc2) . . . . . . . . . . . . . . . . . . . . . . . 161, 175, 253 Recombinant tick anticoagulant peptide (rTAP) . . . . . . . . 51 Recombinant tissue factor pathway inhibitor (TFPI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126–127 Recombinant tissue plasminogen activator (t-PA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57, 69, 91 Renal cell carcinoma. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .123 Reperfusion therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 acute myocardial infarction (AMI) . . . . . . . . . . 34, 39, 91 Restenosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33, 47, 263, 269 animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31, 33 Rivaroxaban acute coronary syndromes (ACS) . . . . . . . . . . . . 194–195 clearance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195–196 clinical trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208 hepatotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196 orthopedic surgery . . . . . . . . . . . . . . . . . . . . . . . . . 166, 196 complications of, 182; venous thromboembolism (VTE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158, 160

rNAPc2, see Recombinant nematode anticoagulant protein c2 (NAPc2) Rose Bengal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32, 54, 60 rTAPr, see Recombinant tick anticoagulant peptide (TAP) r-TFPI, see Recombinant tissue factor pathway inhibitor (TFPI) rt-PA, see Recombinant tissue plasminogen activator (t-PA)

S Selectins Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 human soluble selectin . . . . . . . . . . . . . . . . . . . . . . 263–264 assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263–264 Selenium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 Serotonin release assay (SRA) . . . . . . . . . . . . . . . . . . . 137–140 Serum aminotransferase . . . . . . . . . . . . . . . . . . . . . . see Alanine aminotransferase Shear-induced platelet aggregation . . . . . . . . . . . . . . 2, 17, 20 Sickle cell anemia (SCA) clinical management . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 adult . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296 child . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296 inflammatory mediators . . . . . . . . . . . . . . . . . . . . . . . . . 293 life expectancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 prevalance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 worldwide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 tinzaparin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294 Single-nucleotide polymorphisms (SNPs) cardiovascular disease (CVD) . . . . . . . . . . . . . . . . . . . . 283 definition of . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278 drug metabolizing enzymes . . . . . . . . . . . . . . . . . . . . . . 281 drug transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 hematological disorders . . . . . . . . . . . . . . . . . . . . . . . . . 285 linked to adverse drug reactions . . . . . . . . . . . . . . . . . . 281 mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279–283 warfarin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283–284 SK-BR-3 cell adhesion assay . . . . . . . . . . . . . . . . . . . 270–271 Small animal thrombosis models, 60 SNP, see Single-nucleotide polymorphisms Soluble adhesion molecules . . . . . . . . . . . . . . . . . . . . . 262, 272 as surrogate markers . . . . . . . . . . . . . . . . . . . . . . . . 262, 272 SRA, see Serotonin release assay (SRA) Stasis-induced thrombosis model (Wessler model) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67–69 Stents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209, 214 Streptokinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39, 210 use in animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Synthetic pentasaccharides . . . . . . . . . . . . . . . . . . . . . . . . . 164

T TAP, see Tick anticoagulant peptide (TAP) Tea extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231–232 TEG, see Thrombelastography (TEG) TFPI, see Tissue factor pathway inhibitor (TFPI) TF, see Tissue factor (TF) Thiopurine methyltransferase (TPMT) . . . . . 279, 281–282 THP-1 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22–24 phase-contrast photomicrograph . . . . . . . . . . . . . . . . . . 24 Thrombelastography (TEG) . . . . . . . . . . . . . . . 3–5, 242, 248 Thrombin-induced canine coronary/rabbit femoral artery clot formation . . . . . . . . . . . . . . . . . . . . . . . . . 55–57

ANTICOAGULANTS, ANTIPLATELETS, AND THROMBOLYTICS Subject Index 315 Thrombin inhibitors . . . . . . 5, 37, 44, 47, 54, 58, 66, 71, 89, 142–145, 158, 162–163, 170–173, 183, 188, 196, 235–236 Direct. . . . . . . .32, 54, 71, 142–145, 158, 163, 170–172, 175, 188, 196, 235–236 Indirect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170–171, 235 Thrombocytopenia . . . . . . . . . 72–75, 79, 84, 112, 120, 122, 133–150, 163–164, 171, 173–174, 236, 242, 248–249, 251–252, 254, 269, 302 Cancer . . . . . . . . . . . . . . . . . . 120, 122, 158, 173, 242, 248 heparin-induced thrombocytopenia (HIT) . . . 134–135, 138–141, 149 Thromboembolic disorders . . . . . . . 110, 112, 114, 262, 268 Heparin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118–125 Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . 110, 113–118 Thrombolysis in myocardial infarction (TIMI) trial . . . . . . . . . . . . . . . . . . . . . . . . . . . 194, 208, 214 Thrombolytic agents . . . . . . . . . . . . . . . . . . 38–39, 51, 55, 213 Thrombosis Argatroban . . . . . . . . . . . . . . . . 88, 91, 142, 144, 147–149 factor Xa inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . 181–197 genetic models . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33, 79–88 heparin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .109–127 lepirudin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144–145 low molecular weight heparin (LMWH) . . . . . 148–149 malignancy . . . . . . . . . . . . . . . . . . . . . . . . . . . 162, 241–242 tissue factor pathway inhibitor (TFPI) . . . . . . . . . . . . 162 tissue factor (TF)/activated factor VIIa . . . . . . . . . . . . 187 Thrombosis models animal, see Animal thrombosis models chemically-induced . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 ferric chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 electrically-induced . . . . . . . . . . . . . . . . . . . . . . . . . . . 43–44 Folts, see Coronary thrombosis models Harbauer, see Arterial and venous thrombosis model (Harbauer) laser-induced . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 photochemical-induced . . . . . . . . . . . . . . . . . . . . . . . 59–60 positive controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 rat jugular-vein clamping . . . . . . . . . . . . . . . . . . . . . . . . . 45 in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30–32 wire-coil induced . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60–62 Thrombotic disorders . . . . . . . . . . . . . . . . . . 32, 168, 208, 279 Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280 Tick anticoagulant peptide (TAP) . . . . . . . . . . . . . . . . . 51, 71 Ticlopidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47, 204, 222 TIMI, see Thrombolysis in myocardial infarction (TIMI) trial Tinzaparin (Innohep(tm)) Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121–122, 126 half-life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122–125 metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122–127 pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 sickle cell anemia (SCA) . . . . . . . . . . . . . . . 295, 298–299 acute painful crisis . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 clinical utility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268 Tirofiban clinical benefits . . . . . . . . . . . . . . . . . . . . . . . . 204, 207, 268 combination therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208 heparin-induced thrombocytopenia (HIT). . . . 110, 207 unstable angina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91, 110 Tissue factor pathway inhibitor (TFPI) angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 tissue factor (TF)/activated factor VIIa . . . . . . . . 162 anticoagulant effects . . . . . . . . . . . . . . . . . . . . . . . . 162, 300

biologic actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 cancer . . . . . . . . . . . . . . . . . . . . . . . 110, 122, 127, 159, 255 clinical relevance . . . . . . . . . . . . . . . . . 112, 122, 127, 161, 207, 299 clotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82, 247, 255 deletion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81–82 low molecular weight heparin (LMWH) . . . . . . . . . 110, 112, 122, 126–127, 162, 207, 255, 299 metastasis. . . . . . . . . . . . . . . . . . . . . . . .110, 122, 126–127, 162, 247–248, 251–253 recombinant TFPI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 release . . . . . . . . . . . . . . . . . . . . . . . . 33, 122, 207, 247–248 restenosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 TFPI–2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .162 Thrombosis . . . . . . . . . . . . . . . . . . . . . 82–84, 88, 110, 161 tumor angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . 123, 243 unfractionated heparin (UFH) . . . . . . . . . . . . . . . 247, 255 Tissue factor (TF)/activated factor VIIa Angiogenesis . . . . . . . . . . . 110, 114–115, 120, 123–127, 161–162, 243 anti-TF antibodies . . . . . . . . . . . . . . . . . . . . . 161, 251, 255 as cancer therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 cancer . . . . . . . . . . . . . . . . . . 110, 123–127, 158–159, 162, 242–243, 255 coagulation . . . . . 2, 5, 31–32, 64, 69, 81, 115, 161, 207, 242–243, 281, 293, 299 endotoxemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 factor VII cofactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 gene deletion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 inhibition of . . . . . . . . . . . . . . . . . . . . . . . . . . 110, 115, 126 mediated hypercoagulation . . . . . . . . . . . . . . . . . . . . . . 120 splice variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242 thrombosis . . . . . . . . . . . . . 110, 114–115, 123–124, 127, 158, 162, 255 tissue factor pathway inhibitor (TFPI) . . . . . . . . . 33, 81, 110–115, 122, 207, 247, 281, 299 tumor angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 tumor cell TF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250–251 vascular endothelial growth factor (VEGF) . . . . . . . . . . . . . . 123, 125, 127, 243, 249 venous thromboembolism (VTE) . . . . . . . . . . . . . . . . 159 Tissue plasminogen activators (t-PA) . . . . . . . . . . . . 5–6, 13, 39, 47, 49, 51, 56–57, 60, 69, 80, 82, 84, 87, 91, 111 T-PA, see Tissue plasminogen activators (t-PA) TPMT, see Thiopurine methyltransferase (TPMT) Tumor factors . . . . . . . . . . . . . . . . . . . . . . . . 111, 120, 242–243 predicting sensitivity to anticoagulants . . . . . . . 244, 246 Tumor metastasis . . . . . . . . . . . . . . . . . . . . . . . . . 110, 120, 122, 127, 243 tissue factor pathway inhibitor (TFPI) . . . . . . . 110–111, 115, 122 tissue factor (TF)/activated factor VIIa . . . . . . 100, 184

U UFH, see Unfractionated heparin (UFH) Ulcerative colitis . . . . . . . . . . . . . . . . . . . . . . . . . . 116, 126, 267 Ultrasound/Doppler . . . . . . . . . . . . . . . 41, 44, 46, 48, 52, 55, 60, 62, 206, 213 Unfractionated heparin (UFH) Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 tissue factor pathway inhibitor (TFPI) . . . . . . . . . . . . 112 vs. low molecular weight heparin (LMWH) . . . . . . . . 44

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316 Subject Index

Unstable angina . . . . . 33–34, 39–40, 50, 91, 110, 161, 209, 211–212, 224, 268, 204, 207 tissue factor pathway inhibitor (TFPI) . . . . . . . . . . . . . 33 unfractionated heparin (UFH) . . . . . . . . . . . . . . . . . . . . 11 Urokinase . . . . . . . . . . . . . . . . . . . . . . . . . . 61, 80, 84, 280–281 knock-out mice . . . . . . . . . . . . . . . . . . . . . . . . . . . 73, 82–88

V Vascular cell adhesion molecule-1 (VCAM-1) . . . 111, 114, 265, 272–273 Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272–273 Vascular endothelial growth factor (VEGF) . . . . . 120, 123, 125, 127, 243, 249 tissue factor pathway inhibitor (TFPI) . . . . . . . . . . . . 122 tissue factor (TF). . . . . . . . . . . . . . . . . . . . . . . . . . .121–122 Vascular smooth-muscle cells (VSMC) . . . . . . 124–125, 269 tissue factor pathway inhibitor (TFPI) . . . . . . . . . 33, 81, 110, 207, 247 VCAM-l, see Vascular cell adhesion molecule-1 (VCAM-1) VEGF, see Vascular endothelial growth factor (VEGF) Vena-caval ligation model . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Venous blood collection . . . . . . . . . . . . . . . . . . . . . . . . . . . 9, 18 Venous thromboembolism (VTE) . . . . see also Deep venous thrombosis (DVT); Pulmonary embolism (PE) anti-Xa inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 investigational drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 low molecular weight heparin (LMWH) . . . . . 121–122 major orthopedic surgery . . . . . . . . . . . . . . . . . . . . . . . . 166 fracture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 replacement, 166; hip, 166; knee, 166 NAPc2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161, 175 oral heparin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302–303 prevalence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165, 173 treatment . . 120–122, 158–159, 163, 172–173, 188, 193 acute . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158, 163 long-term . . . . . . . . . . . . . . . . 121, 158, 168, 174, 188 tumor associated VTE . . . . . . . . . . . . . . . . . . . . . . 124–125 unfractionated heparin (UFH) . . . . . 112, 157–158, 302

Venous thrombosis . . . . . . . . . . . . . 31, 47, 61, 65, 67, 81, 84, 118, 146, 163, 168–169, 181–182, 188–189, 193–194, 236, 241, 255, 285 Venous thrombosis animal models, see Animal venous thrombosis models Vessel-wall damage models . . . . . . . . . . . . . . . . 29–32, 64, 66 Viscometric-flow cytometric methodology . . . . . . . . . . . . . 17 Vitamin K . . . . . . . . . . . . . 121, 142, 146–147, 158, 182–185, 195, 229, 284, 286 VLA–1, see Alpha 4 integrin VSMC, see Vascular smooth-muscle cells (VSMC) VTE, see Venous thromboembolism (VTE)

W Warfarin Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 cancer therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182 limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182 natural products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 pharmacogenomics . . . . . . . . . . . . . . . . 279, 283–284, 287 resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208 sickle cell anemia (SCA). . . . . . . . . . . . . . . . . . . . . . . . .292 compared to LMWH . . . . . . . . . . . . . . . . . . 187, 193, 196 Washed platelets (WP) . . . . . . . . 6–9, 12, 18, 23, , 137, 139 isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Wessler model, see Stasis-induced thrombosis model (Wessler model) Wire-coil induced thrombosis . . . . . . . . . . . . . . . . . . . . . 60–62

X Ximelagatran toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158, 167–168 venous thromboembolism (VTE) . . . . . . . 157–161, 163, 165–174 Xylazine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7, 41, 45, 59, 73

Anticoagulants, Antiplatelets, and Thrombolytics (Methods in Molecular Biology)