Effect of heparin–pva hydrogel on platelets in a chronic canine arterio-venous shunt

Effect of heparin-PVA hydrogel on platelets in a chronic canine arterio-venous shunt Cynthia H. Cholakis, Walter Zingg," and Michael V. Seftont Department of Chemical Engineering and Applied Chemistry and the Centre for Biomateriuls, University of Toronto, Toronto, Ontario M5S 1A4 Canada Polyvinyl alcohol (PVA) hydrogel, with or without heparin, was reactive towards canine platelets in a chronic arteriovenous shunt as demonstrated by an increase in platelet regeneration time, a systemic decrease in platelet count and transient decrease in platelet serotonin content. Immobilized heparin (heparin-PVA) had n o effect w h e r e a s unmodified polyethylene was found to be unreactive despite similar levels of platelet deposition as measured by SEM and a higher in vitro reactivity (I. Biomed. Mater. Res., this issue). Twenty-centimeter lengths of hydrogel coated polyethylene tubing were inserted between the arterial and venous portions of the shunt and left in place for 4-6 days, without the complicating artifacts of anticoagulation, anesthesia, or

surgical intervention. Regeneration time was measured as the return to normal platelet cyclooxygenase (co) activity after a single 240-mg dose of aspirin, with co activity measured in uitro as malondialdehyde production. Although measuring new platelet production, regeneration time is an indirect measure of platelet consumption, so that the reduced regeneration time seen here was presumed to reflect enhanced material associated consumption and thromboembolism. Like other hydrogels, PVA does not appear to be "thromboadherent" but it does appear thrombogenic. Immobilized heparin had no additional effect, presumably because the platelet response was dominated by the reactivity of the underlying substrate.

INTRODUCTION

The interaction of heparin, in solution or immobilized, with platelets is unclear. As summarized in a previous article,' both inhibitory and enhancing effects have been noted for heparin either in an aggregometer or by direct assessment of the interaction of heparinized biomaterials with platelets in bead columns or in measurements of adhesion. In the previous article,' we were unable to observe any effect of immobilized heparin, as heparinPVA, on platelets in vitra. Platelet adhesion, release from adherent platelets, and platelet retention were identical on both a heparin-PVA hydrogel and the same hydrogel without immobilized heparin. We continue that study here by reporting on the interaction with platelets, ex vivo in dogs in a chronic AV shunt. *Hospital for Sick Children Research Institute, Division of Surgical Research. 'To whom correspondence should be addressed. Journal of Biomedical Materials Research, Vol. 23, 417-441 (1989) CCC 0021-93041891040417-25$04.00 0 1989 John Wiley & Sons, Inc.

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Heparin was immobilized through its amino acid terminus to a glutaraldehyde crosslinked PVA.* In the absence of significant heparin release, this covalently bound heparin has demonstrated a low in vitro thrombogenicity3-6(partial thromboplastin or thrombin times, thrombin inactivationichromogenic substrate assay, factor Xa inactivation). In addition, in a canine AV shunt the heparin-PVA hydrogel was shown7 to be effective in delaying thrombus formation in 1-mm-ID coated polyethylene tubes at low flow rates (-5 mL/min). The prolonged patency times of heparin-PVA coated tubes with that of control PVA coated tubes without heparin was attributed to the biological effectiveness of the immobilized heparin since the heparin release rate was ?boo of that needed to create a heparin microenvironment.' Here, the same chronic arterio-venous shunt' was used to evaluate the effect of immobilized heparin (and the underlying I'VA substrate) over a 5day period. Previously a parallel flow test section7 was used to expose 1-mm-ID tubes to canine blood at low flow rates, essentially to measure the anticoagulant activity of the immobilized heparin. Here, however, 3mm-ID heparin-PVA (or control) coated tubes were connected in series with the arterial and venous portions of the shunt to expose the entire shunt flow (-180 mL/min) to the coated tube. At this flow rate the shunt remained patent indefinitely provided the shunt material was not too thrombogenic; i.e., it appeared that intrinsic coagulation and fibrin formation were not limiting.

MATERIALS AND METHODS

Heparin-PVA solution Heparin-PVA aqueous solutions were prepared as before,' consisting of 7-10% (w/w) poly-(vinyl alcohol) (99-100% hydrolyzed PVA, J. T. Baker Chem. Co., Phillipsburg, NJ), 5% (w/w) magnesium chloride (Lewis acid catalyst, BDH Chemicals, Toronto, Ont), 0.5-2.0% (w/w) glutaraldehyde (cross-linkingagent, BDH Chemicals), 3% (w/w) formaldehyde (Fisher Scientific Co., Fairlawn, NJ), 4% (w/w) glycerol (BDH Chemicals), 2.0% (w/w) sodium heparin (usually Canada Packers intestinal mucosal, not less than 140 USP U/mg, Toronto, Ont.), and distilled deionized water. Glycerol was added to prevent precipitation of heparin during dehydration. The control hydrogel was prepared in the identical manner but without heparin. The PVA used here was fully deacetylated unlike that used in earlier studies.s7 The PVA was dissolved in double distilled deionized water at -9O"C, followed by cooling of the dissolved polymer to room temperature and then the addition of the other ingredients with extensive stirring. The gel solution was set aside overnight in the refrigerator, to allow entrapped air bubbles to escape and was used within seven days of preparation. Tubes were coated with PVA hydrogel with 2% heparin or 2% "high antithrombin 111 affinity" heparin (34.9 n mole serine/mg, Schering Ag., Berlin). Heparin content of the standard heparin tubes was -15 mg/g

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gel (-16 pg cm') with a release rate of -3 x g/cm2 - min, all based on the toludine blue assay.4 There was a similar glycosaminoglyan content for the "high affinity" heparin surface which was examined in only one experiment.

Coating of polyethylene tubing Polyethylene (PE) tubing (Intramedic, Clay Adams, Parsippany, NJ) was pretreated with 17 M chromic acid (88.5% H,SO,, 7.1% K,Cr,O,, w/w) at 70°C for 8 min and glow discharge cleaned (Harrick plasma cleaner, Ossining, NY) in an air plasma at full power for 45 min. The tubing was then retreated with chromic acid in an identical fashion. This treatment resulted in good adhesion between the hydrogel and the underlying substrate.'' The etched PE tubes (-30 cm long) were coated by filling vertically held tubings with gel solution, followed by draining and drying for 45 min. After four coats were applied, the tubes were left overnight before curing at 70°C for 2 h. Coated tubes were trimmed, reswollen, and stored in PBS.

Chronic arterio-venous canine shunt The AV shunts were made from Silastic medical grade tubing (3.18 mm ID, 6.35 mm OD, Dow Corning, Midland, MI). The shunt was inserted between the iliac artery and contralateral vein, with a short, smaller-diameter Silastic extension (10 cm long, 1.98 mm ID, 3.18 mm OD), used for arterial cannulation. The total shunt length was -100 cm. The shunt implantation is described in detail elswhere.' Briefly, each limb of the shunt, prefilled with heparinized saline (15 IU/mL heparin) was inserted 10 cm into the blood vessel and anchored with grommets to the vessel wall at the cannulation site, and then tunneled under the skin at each side of the body to exit separately from the back of the animal. The animal was held in a snug jacket to prevent damage to the exteriorized portion of the catheter and to minimize reciprocating motion at the wound that could lead to bacterial infiltration and infection. An antibiotic (Pen Strep or Longisil, PVU Inc., Victoriaville, CT, 1 mL daily intramuscularly) and anticoagulant (coumadin, 0.25-0.5 mg/kg orally) were administered postoperatively for the first week only. The shunt flow rate was measured by Doppler ultrasound (Blood velocimeter BV 380 Sonicaid, Fredericksburg, VA). The unit was calibrated with the timed collection of blood in an in nitro peristaltic pump flow circuit.

Insertion of test sections At the start of an experiment, the animal was restrained by hand so that it was sitting comfortably, to enable connection of the 20-cm length of tub-

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ing to the exteriorized portion of the chronic shunt without anesthesia. The test section (test tubing and the connectors) was sterilized in ethylene oxide gas for 3 h, then degassed overnight prior to use. The sterilized tube was filled with sterile saline and the animal's chronic shunt was clamped with tubing clamps (Atraugrip-Doyen Paediatric Intestinal Clamp, Downs Surgical Ltd., England), at both arterial and venous limbs, to stop the flow temporarily. Upon disconnecting the chronic shunt, the test section was quickly fitted onto each limb of the chronic shunt, with the aid of two Silastic external connectors (6.35 mm ID, 9.52 mm OD, 3 cm long) slipped over the ends of the tubing (Fig. 1). After connections were secured, the tubing clamps were removed to expose the test section to the flowing blood. The total occlusion time was -5 min. At the conclusion of the test (up to 6 days) the test section was removed from the main shunt by clamping the shunt once again, and then its two limbs were reconnected with a Silastic connector slipped over the joint. Animals were not used for a new experiment for at least 2 weeks after disconnection to allow all hemostatic factors to return to normal. Blood samples from the shunt, with or without the test section, were taken regularly to measure the platelet count (and other hematological parameters). Platelet counts were determined, in all experiments, by the clinical hematology laboratory of the Hospital for Sick Children in Toronto (ELT-8, Ortho Instruments).

Polyethylenetube Sllastic protector

to vein

Polypropylenecap Silastic connector (635rnmIDx952mmOD)

external portion of chronic shunt

from artery

Figure 1. Schematic illustration of test section showing tubing segment and connectors.

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HEPARIN-PVA HYDROGEL IN A-V SHUNT

Platelet regeneration time A nonradioisotope technique was used to measure the platelet regeneration time during the connection of test segments, to determine albeit indirectly the platelet life span and platelet consumption rate (Fig. 2). The procedure" is based on the observation that a single oral dose of acetylsalicylic acid, aspirin, irreversibly inhibits platelet cyclooxygenase for the life of the platelet. Thus the in vitro thrombin induced peroxidation of platelet lipids is inhibited for those platelets affected by ASA for the life of the platelet. A product of lipid peroxidation, malondialdehyde (MDA), was measured indirectly after reaction with thiobarbituric acid as shown in Figure 2, using a spectrophotometer. The required time for a return to baseline blood production

consumption

platelet count

(CIJ

regeneration time

-

platelet life span

aspirin blocks

cyclooxygenase

Arachidonic acid

1

x7pGG2

J

PGH~-


H

t

PGE., PGF,

etc

HHT

0 Malondialdehyde

( M W

MDA determined as adduct with thiobarbituric acid

(b) Figure 2. (a) Schematic illustration of the difference between platelet regeneration and platelet consumption or life span. (b) Malondialdehyde production as a byproduct of cyclooxygenase activity.

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CHOLAKIS, ZINGG, AND SEFTON

platelet malondialdehyde production following aspirin ingestion was considered the platelet regeneration time. Platelet regeneration times were assayed here by a daily assay of MDA production (nmole MDA/109 platelets) after an oral ingestion of 240 mg of ASA until the pre-ASA value was reached. To collect blood samples, the shunt was disconnected externally and the blood was allowed to flow directly from the arterial cannulae into a blood collection tube containing EDTA (final concentration 1.5 mg/mL) (BDH Chemical Co. Ltd.). Depending on the platelet count, up to 9 mL of blood was required. The citrated blood was centrifuged to obtain platelet-rich plasma, at 150 g for times up to 15 min. In order to minimize the erythrocyte and leukocyte contamination, three spikes to 8009 for 15 s were performed at 5, 10, and 15 min. A platelet count was obtained on this plasma. Platelet buttons were obtained by centrifugation of platelet-rich plasma for 15 min at 1,700g. The supernatant plasma was discarded and the tubes were inverted for 2 min to drain all remaining plasma. The platelet button was resuspended in 0.5 mL of buffered phosphate saline (0.20 M, pH 7.4). Fifty microliters of thrombin (50 U/mL in saline) was added to the samples which were then incubated for 15 min in a 37°C water bath. The platelet suspension was then added to glass test tubes containing 0.5 mL of a 0.53% (w/v) solution of 2-thiobarbituric acid (TBA, Aldrich Chemical Co. Inc.) in a 15.2%perchloric acid (BDH Chemicals).'2After a thorough agitation with a vortex mixer the tubes were heated for 10 min in a boiling water bath. After the tubes had cooled to room temperature, 0.5 mL of 4 N KOH (BDH Chemicals) was added. Subsequently, the tubes were put into the refrigerator for 15 to 60 min. The optical density of the supernatant was then determined at 548-nm wavelength using a Beckman Model 35 Spectrophotometer (Fullerton, CA). A calibration curve was used to convert absorbance to nmole malondialdehyde. Plasma and platelet serotonin assay

To assess the degree of ex v i m activation of platelets during exposure to heparin-PVA and control test segments, the platelet serotonin level was monitored daily. In a few cases plasma serotonin levels were measured also. A spectrofluorometric assay as described by Drummond and Gordon13 was used to determine the serotonin levels. Serotonin was measured by the formation of a fluorophore with o-phthaldialdehyde (OPT). Two milliliters of blood obtained directly from the shunt was added directly to a siliconked test tube containing 0.33 mL of acid citrate dextrose (NIHA)I4and mixed by gentle inversion. The sample was kept and processed at room temperature throughout. Platelet poor plasma (PPP) was obtained in two steps. While citrated blood was first centrifuged at 1508 for 15 min. The platelet-rich plasma was then centrifuged for 5 min at 15,OOOg (Eppendorf

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Centifuge 5412, Brinkman, Westbury, NY). For platelet serotonin content the platelets from a l-mL sample of platelet-rich plasma were recovered. The platelet button (obtained in two steps) was lysed in 1 mL of distilled water. A sample of platelet-rich plasma was also taken for platelet count. The PPP (for plasma serotonin content) and platelet lysates (for platelet serotonin) samples were frozen at -70°C and processed in batches. The samples were thawed just before analysis and vortex mixed. To 0.5 mL of each sample was added 0.5 mL H,O and 200 p L of 100% (w/w) trichloroacetic acid (TGA) (Fisher Scientific Co.) to precipitate proteins. The samples were centrifuged at 3,OOOg for 15 min using an IEC Clinical Centrifuge (International Equipment Co.). The supernatant (0.5 mL) was then transferred to glass stoppered test tubes containing 2 mL OPT/HCl. OPT (o-phthaldialdehyde) HC1 mixture was prepared by dissolving OPT (Sigma) in ethanol (0.5% w/v) and adding 10 volumes of 8 N HCI. The mixture was stored in an amber bottle at 4°C. The mixture was then heated at 100°C for 10 min, and cooled on ice. Excess TCA was removed by extracting with 2 mL chloroform (Caledon Lab, Georgetown, Ont). Fluorescence in the aqueous phase was measured (Model 204 A Perkin Elmer Fluorometer, Norwalk, CT) at an excitation wavelength of 360 nm and an emission wavelength of 475 nm with both slits at 10 mm. Mean blank readings were subtracted from sample readings. A standard curve was constructed and found to be linear up to 300 ng/ mL. Standards were prepared by dissolving 22 mg of serotonin creatinine sulphate (Sigma) in 100 mL deionized distilled water. This stock solution contained 100 pg serotonin/mL. Serial standards from 10 to 300 ng serotonin/mL were prepared. Parbtani and Cameron15have reported the above assay to be sensitive (3 ng/mL), reproducible and unaffected by conditions of takmg or processing the blood. They reported that the concentration of serotonin in plasma or platelets was stable in blood anticoagulated with ACD for up to 6 h at room temperature. Parbtani and Cameron also reported that the standard curve recovered from PPP was identical in slope to that prepared in distilled water with a recovery averaging 100%. Scanning electron microscopy (SEM)

SEM was used to examine the protein and cellular deposits adhering to the tube lumen at the termination of the experiment. The PE tubing was disconnected and the center portion rinsed with -10 mL of isotonic saline in 2-3 s. Segments of the coated tube were fixed in 2% (v/v) buffered glutaraldehyde solution (pH 7.4, 0.008 M phosphate, 0.0015 m potassium, 0.13 M solution) for 48 h. The segments were opened longitudinally, and dehydrated through a graded ethanol series (30,50,70,90,100%) followed by sputter coating (Polaron Sputter Coater, Polaron Equipment Ltd., England) with a 10-nm layer of gold to render it electroconductive and viewed

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in a scanning electron microscope (Hitachi S-520) at an accelerating voltage of 20 KV at the Metallurgy Department, University of Toronto.

In vitro aggregometer studies of heparin potentiation

In vitro enhancement of platelet aggregation by heparin was studied using a Payton Dual Channel Aggregation Module (Payton, Scarborough, Ont). Platelet-rich and poor plasma was obtained by centrifugation of fresh citrated (1 part 3.8% sodium citrate, 9 part WB) canine blood. All tests were conducted at 37°C and at a 1,000 rpm stir rate; no effect of the temperature difference between 37°C and normal canine temperature (39°C) was expected. The PRP was kept at room temperature, and all studies were completed within two hours or blood collection. The reaction mixture contained 0.40 mL PRP, 0.05 mL heparin in 0.154 M NaCl (or saline), and 0.5 mL of the aggregating agent, ADP. Incubation times of PRP and heparin ranged from three to ten minutes. The final concentration of ADP (Aldrich Chemical Company Inc., Milwaukee, WI) was 1, 2, or 5 pM.

RESULTS

Aggregometer studies of heparin potentiation An aggregometer trace showing the enhancing effect of the heparin used in our studies at 10 units/mL on ADP induced aggregation of canine platelets drawn from the shunt is shown in Figure 3. The enhancement was moderate ranging from 4 to 18%, as is typically found for other heparins and human platelets.16 An increase in the concentration of either ADP (5 pm) or heparin did not lead to increases in the potentiation of heparin. Hence it appears that both the heparin and platelets used here are capable of demonstrating the ”expected” in vitro effect. Furthermore the pure bred beagles are ”high responders” using the terminology of Kaplan et al.17who used ADP induced aggregability as a measure of which mongrel dog to use in the study of vascular grafts.

Chronic arterio-venous shunt Animal weights, shunt flow rates and the shunt age at the time of experimentation are reported in Table I for the eight animals used here. The shunt procedure has been successful in providing for chronic patencies as long as 15 months in some cases. We did not observe any effect of shunt age on the parameters examined here, although this has been found a problem in using the parallel flow test ~ e c t i o nwhen ,~ the shunts were older than -8 months. The external connector used to connect test segments as

HEPARIN-PVA HYDROGEL IN A-V SHUNT

425 SpM ADP

i

100 (PPP)

Figure 3. Aggregometer trace showing the sensitivity of canine platelets (from the AV shunt) to ADP (5 pm) and to heparin (10 U/mL). TABLE I Chronic Shunt Variables

Dog

817 830 831 834 835

837 838 849

Weight (kg)

Shunt Flow rate (mL/min)

Age of Shunt at Time of Experiments

15.25 16.00 15.25 13.50 14.75 14.00 13.50 12.50

180 175 190 180 180 175 190 180

11-14 7-8 5-8 1-3 1-5 1-2 1-3 1

(month)

Pretest Platelet Counts (*SD) 389 463 349 269 224

*67 +46 k53 k53 +34

Baseline Malondialdehyde levels (nmoie/1O9plt) 4.72 5.00 5.42 2.97 4.72

-

-

208 +54

3.03

illustrated in Figure 1 was found to be adequate since no animal managed to disconnect the shunt overnight and die by exsanguination unlike the situation with previous connectors. Preliminary short-term experiments were conducted whereby test segments were inserted in the shunt for only a few hours. The addition, in series, of a 20-cm lengths of PE or coated PE tubing of the identical internal diameter as the chronic Silastic shunt did not decrease the shunt flow rate as measured by Doppler ultrasound, except as below. Two of the surfaces, chromic acid oxidized PE and chondroitin sulfate-PVA coated PE were eliminated from further study as their connection to the main shunt resulted in shunt thrombosis. The potential release of thrombi into the circulation posed too serious a risk to the animal. The oxidized PE shunt sections remained patent for 30 and 45 min, in two different animals. The connection of chondroitin sulfate-PVA test segments resulted in shunt occlusion

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in 20 min for two out of six test segments. Connection of the four other test segments resulted in increases in plasma serotonin level after 2 h (increased by 34.5 11.7 ng/mL; &SD, n = 4). For the PVA and heparin-PVA test segments there was found to be no significant increase in plasma serotonin concentration over this time period. Nor was there any decrease in systemic platelet count, over the few hours of exposure.

*

Systemic platelet count (22 days)

A significant decrease in systemic platelet count was noted in seven out of the eight animals with PVA or heparin-PVA coated test segments when exposure was for 2 2 days. Results for the first five animals are shown in Figure 4; these animals had received an aspirin dose 24 h prior to test segment connection to measure regeneration time. In some cases, e.g., dog 835, the decrease in platelet count was substantial and for the safety of the animal the test segments were typically disconnected once the platelet count dropped to 100 x lo9 plt/liter. Although similar trends were seen for both PVA and heparin-PVA, the decrease for PVA was generally not as severe in one or two of the animals. There was no decrease in platelet counts for the polyethylene test segments, in all animals. As expected, no significant difference was observed

600 500 400 300 200 100 $

600

.x, 500

-5

t

831

d34

400 300 - 200 100 0

c

U

600

Time (days)

Figure 4. Systemic platelet counts during test section connection in animals which had received on day 0, a dose of 240 mg aspirin to measure the regeneration time. The test section was inserted on day 1, immediately after removal of day 1 blood sample. control (shunt only, no test section), o polyethylene, A PVA, heparin-PVA.

427

HEPARIN-PVA HYDROGEL IN A-V SHUNT

between the animals which had received a prior dose of aspirin and those which did not, although the latter were only monitored for 2 days. Generally, removal of the hydrogel test segment resulted in a restored platelet count within a week.

Platelet regeneration time The amount of malondialdehyde (MDA) produced per lo9 platelets, 1-5 days after ingestion of aspirin (0-4 days after connection of the test segment) are presented in Figure 5. There is a relatively immediate decrease in MDA production, being
ASA

ASA

Time (days)

Figure 5. Restoration of cyclooxygenase activity of canine platelets (as measured by MDA production as a percent of baseline production) after ingestion of 240 mg aspirin at t = 0, for different shunt materials/animals. Symbols as in Figure 4.

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TABLE I1 Platelet Regeneration Time

Test Shunt Control* Polyethylene PVA Heparin-PVA

Half Time (days) 3.8 ' 0 . 8 2.9 20.3 2.3 k0.2 1.9 '0.3

( n = 4) (n = 4) ( n = 4) (n = 8)

Estimated Consumption Rate ( x lo8, plt/cm2-day) ND 20 215 64 k19 75 t 2 8

*Chronic shunt without any test segment; trx vivo series shunt; 20 cm L; 3.18 m m ID; *SD. n = number of samples tested in 4 or 5 dogs. p < 0.0005 (control vs. heparin-PVA). p < 0.005 (control vs. PVA). p < 0.0005 (control vs. heparin-PVA and PVA). p < 0.0005 (heparin-PVA and PVA vs. PE).

(p < 0.0005) relative to the shunt by itself or to uncoated PE. The difference between heparin-PVA and PVA was not statistically significant. Since the return to baseline levels is indirectly related to the consumption of platelets in the dog with the shunt and test section (the faster platelets are consumed, the faster new platelets are generated), it appears that the level of platelet consumption was greater for the hydrogel containing surfaces (with or without heparin) than for polyethylene. This was also implicit in the drop in platelet counts seen with the PVA hydrogels. The absence of a statistically significant effect of immobilized heparin is discussed below. Platelet serotonin level Platelet serotonin levels were monitored daily to assess (within the normal limitations of this parameter) the degree of platelet activation. As shown, in Figure 6a the connection of PE test segments did not lead to decreased platelet serotonin levels. The platelet serotonin level remained relatively constant throughout the duration of the test. By contrast, the platelet serotonin levels measured each day were more variable during connection of both PVA and heparin-PVA test segments as shown in Figures 6b, c. In five out of six heparin-PVA test segments, a sharp decrease was observed in platelet serotonin concentration after 24 h followed by a partial recovery by 48 h; in the other one (dog 835) there was a steady decrease. The connection of PVA test segments did not lead to the immediate dramatic reduction in platelet serotonin. In three of the animals tested a drop in platelet serotonin concentration was not seen after 24 h but rather became apparent after 48 to 72 h. Daily, during periods of test segment connections, white blood cell, red blood cell, and hematocrit were also measured. No change was detected for all surfaces and with all animals studied.

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days after test segment connection

OPreE21

B2

El3

0 4

3 5

26 2

-

v , 1

c

W

c

a 0

Figure 6 . Platelet serotonin concentration during test segment connection. (a) polyethylene, (b) PVA, (c) heparin-PVA.

Scanning electron microscopy Following 48 h of blood contact the middle section (-2 cm) of rinsed test segments were examined by SEM. Each surface was examined after testing with three different shunted animals (dog 835,838,849). No evidence of red thrombus (i.e., fibrin and red blood cells) nor white blood cells were noted on any of the surfaces. Platelets were seen on all surfaces (Fig. 7) and the number of platelets found varied considerably from surface to surface. No interanimal variability was noted. Isolated platelets, platelet aggregates and fully spread platelet masses could be identified. Pseudopod formation could also be seen. Generally, the hydrogel surfaces (heparin-PVA and PVA) had more fully spread platelets than the polyethylene surfaces. White thrombus formation (i.e., platelet aggregation) was easily detected on both heparin-PVA and PVA surfaces. The polyethylene surfaces had fewer platelets and the platelets were not as clumped together.

Modified heparin-PVA and PVA surfaces Preliminary attempts to reduce the platelet consumption by the hydrogels by immobilizing a greater amount of heparin or a high affinity hepa-

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(4 Figure 7. Scanning electron micrographs of middle portion of tube after 48 h exposure in shunt after rinsing: (a) polyethylene, (b) PVA, (c) heparinPVA.

rin failed, at least as assessed by the drop in platelet count during test segment connection (Fig. 8). Increasing the heparin content via immobilization of a crude heparin with a higher serine content (30.2 vs. 12.2 nmole/mg) did not alter the platelet response. The reduction in systemic platelet count was similar to that for

HEPARIN-PVA HYDROGEL IN A-V SHUNT

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(b) Figure 7. (continued)

the standard heparin-PVA hydrogel surfaces. The higher serine content heparin was obtained from Canada Packers Ltd.; it had been isolated from the mucosa extract but not been purified through the normal series of oxidative steps and solvent fractionations. Immobilization of a purportedly ”high antithrombin I11 affinity” heparin to PVA also resulted in thrombocytopenia during test segment connection.

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(4 Figure 7. (continued)

DISCUSSION

The use of canines The dog is a convenient but by no means ideal animal model for human platelet-material interaction. There are few, if any, alternatives at the

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days after test segment connection

c

50

300

0

5a,

200

I

m -

a 100 n

high affinity crude heparin - PVA - PVA

heparin

Figure 8. count.

Effect of different heparins as heparin-PVA on systemic platelet

moment, for chronic experiments. Canine platelets are generally considered to be more ”reactive” than human platelets, and consequently comparisons of platelet deposition between human and canine blood onto a variety of surfaces show significantly more platelets deposited from canine blood onto most of the surfaces examined.” Lambrecht et al.I9using an ex vivo system, observed greater platelet deposition with canines than with rhesus monkeys also on a number of different surfaces. Furthermore, dogs have been reported to have a low susceptibility to cardiovascular disease and pulmonary embolism and have a very active hemostatic and fibronolytic mechan i s m ~ .These ’~ characteristics have led some researchers to view the canine response as a ”worst case situation” for comparison with human blood-material responses. l8 The fact that canine platelets are similar structurally and functionally to human platelet^,'^-^^ and that canine platelets, like human platelets, are “sensitive” to heparin in solution23supports the hypothesis that the canine model is a reasonable animal model for studying a heparinized material. Such is not the situation for all animals since porcine and bovine platelet^'^'^^ have been reported not to be “sensitive” to heparin. The aggregometer studies confirmed for the canine platelets used here that like human platelets, the platelet aggregating response to ADP was enhanced by the presence of heparin. Other evidence of in vitro heparin sensitivity is that heparin anticoagulation as opposed to citrate anticoagulation on collection of blood samples from the shunt lead to significant reductions in platelet count. In some cases heparin anticoagulation (10 U/mL final concentration) resulted in platelet clumping to the extent that it was impossible to obtain a platelet count. Typically, platelet counts were reduced by -50%, the lower platelet count presumably the result of “spontaneous” platelet activation leading to platelet aggregation.

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CHOLAKTS, ZINGG, AND SEFTON

Canine chronic shunt The AV shunt used here is unique among animal models for studying blood material interactions in that it is a chronic system, free from the surgical artifacts implicit in AV shunts. Without anesthesia, multiple experiments were performed with the same animal, with some animals used for 14 months following surgery. Consequently, interanimal variability, the total number of animals used, and the cost of experimentation were greatly reduced. On the other hand, the shunt does damage the vessels at the cannulation site,’ and this may adversely affect the platelets prior to contact with the test tubing. The progressive effect of this damage does not seem to have been a problem here, since no correlation was found between the age of the chronic shunt and the thrombogenicity of test segments. The advantages of a test system not employing anticoagulants and anesthesia are obvious. For example, pentobarbital, an anesthetic, has been reported to disturb homeostasis by exerting relatively minor effects on cardiac output, arterial pressure, and total peripheral resistance, with more important effects on left ventricular function and myocardial contra~tability.~~ Depression of platelet ADP reactivity bas also been found under the influence of some anesthetic~.’~ In addition, in the absence of surgical trauma, there would be no release of tissue thromboplastin and other endothelial substances. The reason for our success in providing for chronic patencies as long as 15 months is not known. It is presumed that the free floating nature of the 10-cm cannula, fixed with grommets to the tissue rather than ligated directly to the vessel, allowed for a less traumatic flow pattern (perhaps a reduced degree of turbulence or a reduction in unavoidable stagnation zones) with consequently less or delayed thrombosis.

Ex vivo platelet interactions with PVA hydrogels It is clear from the results that, regardless of the presence or absence of heparin, the PVA hydrogel is reactive towards platelets: platelet counts decreased, serotonin levels decreased (at least temporarily) and platelet regeneration times increased in the presence of a 20-cm length of 3-mm-ID PVA coated tubing in the AV shunt. Not surprisingly no evidence could be found for large numbers of aggregates either by the Wu and Hoak methodz6 or from the platelet volume distribution as measured as part of the platelet count determination. The scanning electron micrographs at 48 h of blood contact support this view with more cells and more spread cells on hydrogel surfaces than on polyethylene surfaces. The approximate number of platelets on the hydrogel surfaces after 48 h ex v i m exposure was -20/1000 pm2, which is comparable to the adhesion density (but not the degree of spreading) seen in vitro in a Chandler loop.’ Interestingly in vitro polyethylene had more adherent platelets than the hydrogels, whereas ex vivo the reverse was true.

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Whether this reflects an inadequacy of in uitvo tests as a prediction of ex uivo performance, a difference in observation times (1 h or 2 days), a specific problem of the air interface in the Chandler loop or is a peculiarity of polyethylene is a question for further study. The degree of platelet adhesion observed here or in vitvo for the hydrogels is not particularly high (relative to highly thrombogenic surfaces e.g., fibrinogen coated glass or subendothelium). On the other hand, the degree of platelet consumption implicit in the decrease in the platelet count or in regeneration time is very high. Thus it appears that platelets contact the hydrogel surface but do not remain adherent. This transient contact however is enough to alter the cell, perhaps make it more sensitive to aggregation, so that it aggregates in the bulk fluid and/or is removed from the circulation earlier than normal. Alternatively platelets may aggregate at or near the surface, perhaps in the presence of a ”fog” of released substance~,’~ without the aggregates attaching to the surface. Thus the observation of few adherent platelets implies that the surface is not ”thromboadherent” but the surface is not nonthrombogenic. A similar conclusion was reached by Hanson et a1.” who demonstrated a direct linear relationship between the water content of hydrogels (eight grafted acrylic polymers and copolymers) and the rate of platelet consumption in a baboon AV shunt. Although they did not test PVA, their results suggested that hydrogel surfaces are consumptive toward platelets, even though few platelets adhere to the surface.

Effect of immobilized heparin The results reported here support our hypothesis’ that immobilized heparin, a t least in the form of heparin-PVA, has no effect on platelets. Rather the dominant interaction is that of the substrate, here PVA, on platelets, and ex viuo PVA is reactive toward platelets. Ex vivo immobilized heparin had no significant effect, relative to the PVA without heparin, on the systemic platelet count decrease, the regeneration time decrease, the platelet serotonin decrease or the SEM views of the surface. A similar conclusion was presented earlier on the basis of in vitro measurements of platelet adhesion, release and, retention.’ However, there may yet be an adverse effect of the immobilized heparin that the experiments performed to date have not been able to detect. It should be noted that in each experimental situation, where a difference, albeit statistically insignificant, has been observed, the heparin PVA is always worse than the PVA: regeneration time was slightly lower, platelet release was greater, platelet counts decreased slightly faster, bead column retention was slightly greater. Unfortunately, it is not possible to combine these three observations to improve their statistical significance. Nonetheless, it is clear that the immobilized heparin in the PVA hydrogel has only a small effect (if any) on platelets, at least for the relatively small areas of material, and the low heparin surface content. Although the surface heparin concentration

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may be an important factor, the heparin concentration is sufficient to accelerate the inactivation of thrombin and prevent fibrin formation. Hence in a practical sense, the effect of heparin on platelets may be neglected. Increasing the surface heparin content by using a crude heparin or increasing its effectiveness against antithrombin I11 by using a ”high affinity” heparin had no effect, but the result must be considered preliminary. It still remains to be seen what happens with higher surface areas of material, more highly heparinized surfaces or with less reactive substrates. The difference between the effect of heparin seen here and the enhan~ed~’-~l or r e d ~ c e d ~aggregation ’,~~ seen by others may be attributed to the usual factors in biomaterial evaluations: different species, methods of investigation or heparinization technique, and the difference between in vitro and ex vivo results. However, the most important factor may be the inadvertent absence of suitable controls in several cases, since the observed effect may have been attributed to the presence of heparin, even though the modified surface without heparin was not tested. Platelet serotonin level Although monitoring platelet serotonin was useful as an indicator of release and hence provided independent support for the observation of reduced regeneration time, these results are not unequivocal since platelets are known to re-uptake the released serotonin. Hanson and Harkerx have suggested about 10% of the released serotonin is reutilized based on simultaneous measurements of platelet survival in a baboon model with 51Crand 14 C-serotonin. The platelet serotonin data presented in this thesis suggest that, especially for heparin-PVA, extensive release, greater than 50%, occurred during the first 24 h. It is presumed that subsequently, the production of new, heavier platelets restored partially the serotonin level since younger platelets have more granular structures (i.e., serotonin) than older platelets.35 Platelet regeneration time The daily determination of platelet MDA production (nmole MDA/ 10’ plt) before and after one single intake of aspirin was found to be a reliable technique for the assessment of platelet regeneration times in canines. Compared to platelet survival time determinations using radiolabels, platelet regeneration time measurements are safer and less invasive. The assay required no exposure to radioactivity, no initial withdrawal of large amounts of blood and no reinjection of manipulated platelets. The platelet regeneration time test was found to be a test applicable to repeat studies within a limited time period. The primary disadvantage of this method relates to aspirin’s effect on platelet behavior. By irreversibly inhibiting cyclooxygenase, platelet production of PGG,, PGH2, and thromboxane A, are

437

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inhibited and hence those platelets will not aggregate. However, the aggregability of human platelets is restored within a few hours of aspirin ingestion (650 mg), presumably reflecting the entry into the circulation of new platelets (as little as 2.5% aspirin-free platelets) with cyclooxygenase activity that has not been affected by aspirin.36Nevertheless, it is conceivable that the aspirin-inhibited platelets may interact with the bioinaterial surface differently than native platelets, to reduce the apparent platelet consumption. Harker and H a n s ~ n , ~ however, ' reported that in baboons the oral administration of 10 to 330 pmol/kg/day (equivalent to 270 to 900 mg ASA/day for 14 kg canines-not a single one time only dose) had no measurable effect on platelet consumption by tubes inserted in a chronic AV shunt. A similar conclusion is presumed true for heparinized surfaces and hence we presume that aspirin-inhibited platelets are not consumed differently relative to aspirin-free platelets on such surfaces. Another disadvantage is the lower sensitivity of the MDA method relative to the use of "'In. Larger blood samples are needed to compensate and thus it is almost impossible to obtain more than one data point per day. Aspirin also inhibits vascular endothelium production of prostacyclin, but this effect is transient and not likely to affect the consumption of platelets over the 4-5 days of the experiment. Similarly the inhibition of platelet cyclic endoperoxide production and the absence of these for use by vascular endothelium (cell-cell "steal" pathway) is not expected to affect the regeneration time, at least in comparing various materials. Platelet regeneration times are not biologically identical to platelet survival times, rather they are complementary parameters. The former assesses the appearance rate of new platelets into the circulation while the latter measures the disappearance rate of circulating platelets. Roncucci et aL3' reported that patients with synthetic valves or vascular prostheses (Starr and/or Bjork valves and aorto-iliac Dacron grafts) have clearly decreased platelet regeneration times, in quantitative agreement with measurements of platelet consumption and turnover. The original MDA method" was shown to give identical results in terms of lifespan and turnover rates to those obtained with radiolabelled platelets in individuals without biomaterial implants. The effect the chronic shunt itself has on platelet regeneration time is unknown since we were unsuccessful in taking blood samples atraumatically from the dogs without shunts. It is likely that it involves a minor, if any, decrease in platelet regeneration time. Human platelets normally survive for 8 to 10 days in the circulation whereas Porter-Fink et al.39reported a platelet survival time in six normal dogs of 6.3 0.7 days utilizing indium-lll-8hydroxyquinoline as the platelet label. Rauls et al.40reported a mean platelet survival time using "'In for beagle dogs of 5.4 f- 0.53 days, fitting the data to a linear model. Wilkinson et aL41demonstrated that platelet survival in beagle dogs was linear with a 13.6% loss per day, giving a total survival time of 6.9 5 0.24 days. Similar numbers are expected for regeneration times. Roncucci et al.38reported, in human volunteers, that the mean PRT half-time was estimated at 4.0 k 0.3 days. Since dog platelet life spans

*

438

CHOLAKIS, ZINGG, AND SEFTON

are less, our MDA Ievels returned to the baseline levels in >5 to 6 days and the mean PRT half time was 3.8 2 0.8 days suggest that the shunt did not substantially affect the regeneration time. It is clear from the regeneration time results that the presence of the hydrogels increased the consumption rate of platelets to the extent that the rate of production of new platelets had to be increased to compensate (and this was insufficient given the platelet count decrease). However, it is not possible to determine a precise platelet consumption rate from the raw data since the assay measures the increase in the percentage of circulating platelets unaffected by aspirin and not the number of new platelets brought into the circulation. Unfortunately, we cannot estimate what fraction of the new platelets are removed prior to our MDA measurement. Nevertheless if one assumed that over the first 24 h, there is no removal of the new platelets, then it is possible to estimate a consumption rate. These rates are gwen in Table 11. In contrast in baboons, Hanson” found polyethylene and polydimethylsiloxane to be the least consumptive of all materials tested (2 X lo8 pIt/cm2-day)with Biomer and polyacrylamide (grafted into Silastic) to be the highest at 19.8 and 18 x 10’ plt/cm2-day. Presumably the difference in consumption values represent in large part species differences, although this remains to be tested. Unfortunately neither the effect of the shunt itself nor normal senescence can be subtracted from these calculations. Finally it must be noted that the detailed sequence of events that occurs once a platelet has contacted a surface in such a shunt and is eventually removed from the circulation is largely unknown. Unlike adhesion or aggregation, plateIet consumption is more difficult to follow since most of the effect occurs after the platelet has left the surface. Also the clinical significance of this phenomenon (in the absence of thrombocytopenia) is unclear since it is not known whether consumption represents microemboli formation or not. These questions and others remain to be answered. CONCLUSIONS

The ex Z ~ Z D O series shunt has been found to be an excellent system for evaluating the effect of heparin-PVA and PVA hydrogel on platelets. It is presumably useful for testing other potentially compatible materials. It allows for repeated testing in conscious dogs without anesthesia and without surgical artifacts. Consequently, interanimal variability, the number of animals and the cost of experimentation are reduced. The tests employed are sensitive to the surface properties of test segments. In this shunt, in canines, the PVA hydrogel was reactive towards platelets, and the presence of the immobilized heparin had no significant effect on that reactivity. Connection of heparin-PVA and PVA tubing segments (20 crn long, 3.18 mm ID) resulted in substantial decreases in systemic platelet counts (thrombocytopenia). Reductions in counts were approximately 50% over a 48-h period. There was also a greater than 50% reduction in platelet serotonin levels 24 h after connection of heparin-

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PVA tubing segments. Finally, platelet regeneration times were reduced (50% decrease in mean half time) during the connection of heparin-PVA and PVA test segments indicating an increase in the platelet consumption rate. Despite the ability of heparin-PVA to inhibit fibrin formation it was found to be reactive towards platelets. However, it appeared that the substrate, PVA, dominated the platelet response and that the presence of heparin per se did not alter the situation. In vitro platelet adhesion and release of adherent platelet values reported previously were not a good indicator of ex vivo performance. While the conclusion about the absence of an effect of immobilized heparin is unchanged, the platelet reactivity of PVA was not predicted on the basis of the previously reported in vitro results.' Hence the significance of in vifro measurements as a predictor of ex vivo performance is questioned. The authors acknowledge the financial support of the NIH (HL 24020) and a scholarship (CHC) from the Natural Science and Engineering Research Council of Canada. We are also grateful for the technical assistance of Wan Ip in the animal experiments.

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Received June 28, 1988 Accepted July 27, 1988