CTAP-III, TG, and NAP-2 Ernst Brandt*, Andreas Ludwig and Hans-Dieter Flad Research Center Borstel, Center for Medicine and Biosciences, D-23845 Borstel, Germany * corresponding author tel: 49-4537-188444, fax: 49-4537-188404, e-mail:
[email protected] DOI: 10.1006/rwcy.2000.10005.
SUMMARY CTAP-III, TG, and NAP-2 are proteolytic truncation products derived from a common precursor (PBP) that is synthesized in megakaryocytes and secreted by activated platelets. NAP-2 is generated by enzymatic processing by leukocytes and is active on neutrophils, affecting chemotaxis, degranulation, adhesion receptor expression, and respiratory burst via high- and low-affinity interaction with chemokine receptors CXCR2 and CXCR1, respectively. Its precursor CTAP-III also affects metabolic activities in connective tissue cells by as yet unknown mechanisms. In addition to their proinflammatory and reparative functions, these predominantly intravascularly localized chemokines appear to exhibit proinflammatory roles. This is indicated by their modulatory effects, e.g. on megakaryocytopoiesis, on endothelial prostacyclin secretion, on basophil leukocyte histamine release, and even on T cell cytotoxicity.
BACKGROUND
Discovery The CXC chemokines, platelet basic protein (PBP, 94 amino acids), connective tissue-activating peptide III (CTAP-III, 85 amino acids), -thromboglobulin ( TG, 81 amino acids) and neutrophil-activating peptide 2 (NAP-2, 70 amino acids) are homologous and immunologically crossreactive proteins. Their
amino acid sequences only differ in the length of their N-termini. TG was discovered in 1975 as a product released by thrombin-activated platelets (Moore et al., 1975), and its complete primary structure was reported 3 years later (Begg et al., 1978). The discovery of CTAP-III dates back to 1977, when it was purified from human platelets and reported to stimulate hyaluronic acid formation and DNA synthesis in human connective tissue cells (Castor et al., 1977). Subsequently CTAP-III was found to be identical to a secreted platelet protein termed lowaffinity platelet factor 4 (LA-PF4) and to represent a precursor of TG (Niewiarowski et al., 1980). According to the latter authors, conversion of CTAP-III into TG occurs following its release from activated platelets and is catalyzed by a plateletsecreted protease. PBP, a highly cationic protein (pI 10) isolated from platelet release supernates in 1980, was reported to be mitogenic for Swiss 3T3 mouse cells (Paul et al., 1980), but its relationship to CTAPIII and TG became clear only 6 years later, when it was recognized to represent an N-terminally extended precursor of the former proteins (Holt et al., 1986). NAP-2, the smallest protein within this series, does not originate directly from platelets, but was discovered almost simultaneously in 1989 by two different groups as a cleavage product formed in supernates of platelet-containing peripheral blood leukocyte cultures (Brandt et al., 1989; Walz and Baggiolini, 1989). Following the discovery of NAP-1 (now known as IL-8), NAP-2 represents the second chemokine to become recognized as a neutrophilactivating mediator, stimulating functions like
1078 Ernst Brandt, Andreas Ludwig and Hans-Dieter Flad lysosomal enzyme degranulation (Walz and Baggiolini, 1989), Fc receptor-dependent phagocytosis (Brandt et al., 1989), and as shown later, directed chemotactic migration (Walz et al., 1989).
sheet arranged in a Greek key fold, and by one helix at the C-terminus (Figure 1). Oligomer formation, depending on the individual molecular species and the physicochemical conditions, has been observed.
Alternative names
Main activities and pathophysiological roles
Due to their immunological crossreactivity with polyclonal antisera PBP, CTAP-III, TG, and NAP-2 are also collectively termed TG or TG antigen. Because of its structural similarity to platelet factor 4 (PF4) and its lower capacity to bind heparin, CTAP-III is sometimes referred to as low-affinity platelet factor 4 (LA-PF4). Initially an alternative name for NAP-2 was factor C; however, this is no longer in use.
Structure PBP, CTAP-III, TG, and NAP-2 are small proteins, their molecular weights ranging from about 10 kDa to 8 kDa, depending on the degree of N-terminal truncation of the individual molecules. These proteins share the basic structural features found in all other CXC chemokines, i.e. they contain four cysteine residues at conserved positions, and the two cysteines most proximal to the N-terminus are separated by one amino acid residue, which in TG antigen proteins is a methionine. In all the TG antigens the functionally important tripeptide sequence Glu-LeuArg (ELR motif), located directly upstream from the CXC motif is retained. The overall tertiary structure of these molecules corresponds to that of other CXC chemokines, comprising an extended N-terminal loop, followed by three strands of antiparallel
Both PBP and CTAP-III have been reported to exhibit growth factor-like activities on connective tissue cells, although the mitogenic properties of these proteins remain controversial. CTAP-III has however been shown to stimulate various aspects of metabolism in connective tissue cells, including synthesis of matrix components and glucose uptake. TG was found to be devoid of these activities, while NAP-2 was reported to be functional. In contrast with its precursors PBP, CTAP-III, and TG, which are practically inactive in this respect, the main physiological role of NAP-2 is that of an activator of neutrophil effector functions. These include chemotaxis, degranulation, upregulation of adhesion molecules, and others. However, PBP and CTAP-III may downmodulate NAP-2-induced functions, as specified below. Potential pathophysiological roles of TG antigen proteins are only beginning to emerge. In general elevated plasma levels of TG are indicative of platelet activation and are found in patients with e.g. peripheral vascular disease, diabetes mellitus and coronary artery disease. Corresponding findings were reported in patients suffering from rheumatic disease and renal disease. In these patients CTAP-III deposition in the synovium and kidney, respectively, was associated with partial conversion into NAP-2, suggesting the involvement of these chemokines in pathological neutrophil and connective tissue activation.
Figure 1 Basic secondary structure of TG antigen proteins. The most prominent structural features of NAP-2 (amino acid positions 1±70) and its N-terminally extended precursors PBP, CTAP-III, and TG (starting at positions ÿ24, ÿ15, and ÿ11, respectively) are shown. The four conserved cysteine residues and the mode of disulfide bridging are indicated in blue and the position of the ELR motif is highlighted in red. The three sheet strands and the C-terminal helix are symbolized by zig-zag lines and a cylinder, respectively (secondary structure information according to Malkowski et al., 1995). (Full colour figure can be viewed online.)
CTAP-III, TG, and NAP-2 1079
GENE AND GENE REGULATION
Accession numbers TG1 (PBP) gene: PIR A39546 (Majumdar et al., 1991) CTAP-III cDNA: GenBank M11517 (Mullenbach et al., 1986); M54995, M38441 (Wenger et al., 1989); PIR A37382 (Wenger et al., 1989)
Chromosome location The gene encoding human PBP, which is the parent molecule of the proteolytic derivatives CTAP-III, TG, and NAP-2, is located on chromosome 4. It maps to locus 4q12-q13 as a duplicate (termed TG1 and TG2) and occurs in multiple copies (Majumdar et al., 1991; Wenger et al., 1991). According to one report (Zhang et al., 1997), the TG2 is a nonexpressing pseudogene.
Figure 2 Structure and organization of the PBP gene ( TG1). The three exons are shown as boxes, and within the exons the locations of further segments representing untranslated regions (light gray), the region coding for the leader sequence (dark gray), and those coding for the mature protein (black) are indicated. Numbers within these segments stand for the numbers of base pairs. Upstream from the commonly used transcriptional start codon (position 1) there is an alternative site at position ÿ4. The locations of the regulatory elements (PU box and TATA box) are given in green and blue, respectively. Data are according to Zhang et al. (1997) and Majumdar et al. (1991), except for the segmentation of exon 1. The latter authors, using a TG cDNA, assigned 141 bp to the leader sequence, while according to Wenger et al. (1989), using the longer PBP cDNA, the leader sequence comprises only 102 bp. (Full colour figure can be viewed online.)
Relevant linkages Besides the PBP genes TG1 and TG2 the chromosomal region 4q12-q13 contains the genes for several other members of the CXC chemokine family, including those for PF4, IL-8, and MGSA/ GRO, GRO , GRO , but not IP-10 (Wenger et al., 1991; Tunnacliffe et al., 1992). The former genes are closely linked to form a cluster on a single 700 kb Sfil fragment. Especially close linkage of the PBP genes exists with that for PF4, which also occurs as a duplicate (PF4 and PF4alt, respectively). While TG1 and PF4 are separated by < 7 kb, the distance between TG2 and PF4alt is approximately 5 kb, and within each TG/PF4 duplication the TG-like gene is upstream of its linked PF4-like gene (Tunnacliffe et al., 1992).
Regulatory sites and corresponding transcription factors The gene for PBP ( TG1) was first cloned by Majumdar et al. (1991) using a TG cDNA probe isolated from a cDNA library derived from the megakaryoblastic cell line DAMI (Majumdar et al., 1991). The gene was found to have a total length of 1139 bp and to be divided into three exons (Figure 2). In this respect the gene was similar to that for PF4 (three exons), but differed from those for other
chemokines, including IL-8, the MGSA/GRO subfamily, and ENA-78, which all contain four exons. Exon 1 consists of an 87 bp 50 UTR, followed by 102 bp coding for the signal peptide. The remaining 46 nucleotides encode the N-terminus of the mature PBP. Exon 2 is 136 bp long and exon 3 contains the remaining 100 bp of the coding region, including the stop codon (TAA). The rest of exon 3 consists of a 218 bp 30 UTR. There are two regulatory sites upstream from the more commonly used transcriptional start site, an alternative one being located at ÿ4 bp. A canonical TATA box is located at ÿ32 bp to ÿ25 bp (Majumdar et al., 1991). A second regulatory element within a pyrimidine-rich tract is located at ÿ78 bp to ÿ75 bp and was found to match a PU box as well as to bind the hematopoietic transcription factor PU.1, that belongs to the Ets family (Zhang et al., 1997).
Cells and tissues that express the gene Platelets are by far the major source for mature TG proteins, but exhibit only marginal mRNA expression. They are storage sites for PBP and CTAP-III, while gene expression and protein synthesis occur during maturation of the precursor cells, the megakaryocytes. More recently, leukocytes of the
1080 Ernst Brandt, Andreas Ludwig and Hans-Dieter Flad myeloid and lymphoid lineage have also been shown to express the gene (Table 1).
PROTEIN
Accession numbers PBP: PIR A24448 (Holt et al., 1986) CTAP-III: PIR A93982 (Castor et al., 1983), MIM 121010, SwissProt P02775 TG: PIR A90411 (Begg et al., 1978) NAP-2: PIR PL0222 (Walz and Baggiolini, 1990)
Sequence
34-residue leader sequence. The mature PBP contains 94 amino acids and constitutes a precursor of further N-terminally truncated derivatives, the main representatives being CTAP-III (85 amino acids), TG (81 amino acids), and NAP-2 (70 amino acids). Further truncation products have been found, as described below. All proteins contain four cysteine residues (positions 5, 7, 31, and 47) that are conserved in all CXC chemokines, and N-terminal to the first cysteine the ELR-sequence motif, that is present in all CXC chemokines capable of interacting with chemokine receptors CXCR1 and CXCR2.
Description of protein Physicochemical Properties
As shown in Figure 3, PBP is translated as a 128 amino acid precursor protein (pre-PBP), bearing a
Like other chemokines, TG proteins are relatively resistant to denaturing conditions, such as low pH,
Table 1 Human cells and cell lines expressing the PBP gene Cell type
Remarks
Reference
Megakaryocyte
Major gene-expressing cell type, expression during maturation
Evidence derived from cell lines/platelets (see below)
Platelet
Low level of mRNA expression, (probably not regulated)
Wenger et al., 1989; Power et al., 1995
DAMI (megakaryoblastic cell line)
Constitutive expression
Majumdar et al., 1991
HEL (erythroleukemic cell line)
Constitutive expression
Zhang et al., 1997
T cells
Induction by PHA/PMA
Skerka et al., 1993
Monocytes
Induction by LPS
Iida et al., 1996
Neutrophil granulocytes
Induction by LPS
Iida et al., 1996
Figure 3 Sequences of TG proteins. Numbers indicating the position of amino acids relate to the mature NAP-2 (residues 1±70). Thus the sequence of pre-PBP starts at position ÿ58, containing the leader sequence (shown on top), the N-terminus of mature PBP (positions ÿ24 through ÿ14), of CTAP-III (positions ÿ15 through ÿ12), of TG (positions ÿ11 through ÿ1), and of NAP-2. The respective N-terminal residue in the precursor proteins is shown in blue, the location of the ELR motif is highlighted in red, and the four conserved cysteine residues are shown in yellow. (Full colour figure can be viewed online.)
ELR C C
C
C
CTAP-III, TG, and NAP-2 1081 high salt, and elevated temperatures. This is probably due to the presence of two disulfide bridges connecting Cys5 to Cys31 and Cys7 to Cys47, thereby stabilizing the overall conformational structure. Reducing agents lead to unfolding of the proteins and loss of biological activity. All of the TG proteins show medium affinity for heparin, which may be due to the clustering of positively charged amino acids at certain regions of the molecules, especially at the Cterminus. However, the positive net charge found in most of the proteins (See Table 2) does not appear to represent the only characteristic responsible for heparin binding. In contrast to its close relative PF4, the tendency of TG proteins to form oligomers (dimers and tetramers) is low and decreases with the extent of N-terminal truncation (Yang et al., 1994). At physiological concentrations (nanomolar) NAP-2 generally exists in a monomeric state, while the Nterminally extended precursors, which are released at concentrations in the micromolar range show some dimer formation (Table 2). Secondary Structure The solution structure of monomeric NAP-2 (Mayo et al., 1994), as well as the crystal structure of the
tetrameric chemokine (Malkowski et al., 1995), have been reported. The recombinant NAP-2 proteins analyzed by these authors were slightly different from the native NAP-2, in that the former (Mayo et al., 1994) used a 72-residue molecule bearing an additional Met at the N-terminus and a Tyr at the C-terminus, while the latter (Malkowski et al., 1995) used a 70-residue molecule having the Met at position 6 exchanged for Leu. The basic secondary structure data for monomeric and oligomeric NAP-2 are in good accordance, consisting of a long N-terminal open loop, three strands of sheet, followed by an helix at the C-terminus. However, some minor divergence was found in the exact location of these structural elements as well as of additional ones residing within the N-terminus and the connecting loops (Figure 4). According to Mayo et al. (1994) strands 1, 2, and 3 encompass residues K21±K27, V34±I40, and R44±D49, respectively, these elements are allocated by Malkowski et al. (1995) to residues I19±G26, V34±T39, and R44±L48, respectively. The C-terminal helix is reported to contain 13 residues (R54±D66) by the former authors, while it is 12 residues long (I55±D66) according to the latter authors. However, both groups find that the ultimate
Table 2 Physicochemical properties of TG proteins Property
Protein PBP
CTAP-III
TG
NAP-2
Chain length (amino acids)
94
85
81
70
Molecular weight (Da)
10,262
9288
8861
7624
Isoelectric point (pI)
10
8
7
8.8
Number of charged residues (cationic/anionic)
17/13
14/13
13/13
12/9
Monomer/dimer ratio at nanomolar concentrations micromolar concentrations
2000/1 1000/1
500,000/1 5000/1
Figure 4 Secondary structure of NAP-2. An allocation of secondary structural elements to the primary sequence of NAP-2 as reported by Mayo et al. (1994) (above) and Malkowski et al. (1995) (below) is shown. Stretches of sequence that are organized into strands and helices are highlighted in green and yellow, respectively, while turns within the N-terminus and the connecting loops are boxed in red. Amino acids shown in blue represent residues that were introduced (above) or replaced (below) in the recombinant proteins for experimental reasons. (Full colour figure can be viewed online.)
1082 Ernst Brandt, Andreas Ludwig and Hans-Dieter Flad four residues of NAP-2 (E67±D70) are not integrated into the helix, but form fraying ends of undefined structure. Tertiary Structure and Subunit Association The tertiary structure of NAP-2 as derived from its four-subunit crystal structure described by Malkowski et al. (1995) is essentially consistent with that of the monomeric chemokine in solution described by Mayo et al. (1994). Both groups agree on the presence of a long N-terminal loop, a triplestranded antiparallel sheet arranged in a `Greek key' with strand 2 hydrogen-bonded to strands 1 and 3. The N-terminal open loop containing the ELR sequence motif is stabilized by the two disulfide bridges and by two hydrogen bonds running from T11 to C47 and D49, respectively. Additionally the hairpin loop connecting strands 1 and 2 contains a turn that is folded proximal to the ELR motif. The strands 2 and 3 are also connected by a hydrogenbonded hairpin loop, while a loop containing a single turn leads into the C-terminal helix that runs diagonally across the top of the sheet. As derived from the crystal structure of the tetrameric molecule, the latter is made up of two pairs of dimers (A/B, C/D) each forming an extended -pleated sheet of six strands, which are arranged back to back to form the tetramer. The forces stabilizing the association of monomers into dimers (e.g. for dimer A/B) are contributed mainly by hydrogen bonding of residues Q20A and G26B, L22A and V24B, V24A and L22B, as well as G26A and Q20B, which are located within the strands 1 of the opposing subunits. Further dimer stabilization is contributed by hydrophobic clustering of several residues located within the two helices that extend over the sheet of the respective adjacent subunit. Within the same subunit I55A and I58A from the helix make contact with, e.g. I14A and P16A residing in the loop connecting strands 1 and 2. The helix±sheet interface of the AB-dimer is further stabilized by contacts between helix residues V59A, K62A, and L63A, and sheet residues L22B, V24B, and V36B (Figure 5).
Discussion of crystal structure As already mentioned, the tendency of NAP-2 to form tetramers and dimers under physiological conditions is extremely low. In all likelihood the tetrameric crystal structure described by Malkowski et al. (1995) results from the experimental conditions required for growing crystals (high protein concentration, pH 4.6) and may also be due to the replacement
Figure 5 Tertiary and quarternary structure of NAP-2. A schematic overview of the topology of a NAP-2 A/B-dimer. Single or double hydrogen bonds are indicated by singleand double-headed arrows (going from the donor to the acceptor), respectively. Residues within strands 1, that contribute hydrogen bonds to the dimer interface are circled in red. The residues that form hydrophobic clusters in one monomer, and those that contribute to stabilizing the dimer are highlighted in yellow and green, respectively. For reasons of clarity, only the corresponding residues from one of the monomers are shown. (Full colour figure can be viewed online.)
of the naturally occurring residue Met at position 6 for Leu in the recombinant NAP-2 used in these studies. The related chemokine PF4, having a Leu at the homologous position, readily forms tetramers. In functional terms, neither tetramers nor dimers are required for NAP-2 biological activity (Rajarathnam et al., 1997). The reduced ability of NAP-2 to oligomerize may also be responsible for its lower heparin binding affinity as compared with PF4. Although the crystal structures of the N-terminally extended precursors PBP, CTAP-III, and TG have not been reported, analyses of a NAP-2 molecule extended by five N-terminal residues (i.e. CTAP-III [des 10]; Malkowski et al., 1997) suggest that the overall secondary tertiary structure of the NAP-2 portion in these molecules is not changed. Nevertheless, these precursor molecules have a higher potential for oligomerization and are not neutrophil stimuli. Both phenomena may be related, since it was found that the N-terminal extension in CTAP-III [des 10] is not disordered, but forms a unique structure as compared with equivalent residues in other chemokines. By
CTAP-III, TG, and NAP-2 1083 folding back to interact with the ELR motif, a type II turn is formed by residues L2 through E2, giving rise to further stabilizing interactions (salt bridges) between residue E2 and two arginines (R4/R44) within the same subunit. This folding leads to masking of the ELR motif and may therefore be responsible for lack of receptor binding of N-terminally extended precursors. It may also have implications for oligomerization, as it helps to establish novel intersubunit interactions, e.g. H-bonds between L3B±K45D and L3D±K45B. These interactions also help to immobilize the ELR region.
Important homologies The NAP-2 portion of TG proteins displays relevant structural homology to other CXC chemokines of both the ELR and non-ELR subfamily, while the Nterminal extensions upstream from the ELR motif (as found in TG, CTAP-III, and PBP) are unique in sequence and spatial arrangement (Figure 6). With respect to ELR chemokines, highest sequence
similarity exists to MGSA/GRO (41 identical amino acids, homology: 59%) and IL-8 (33 identical amino acids, homology: 47%). The closest relative among nonELR chemokines is the PF4 (37 identical amino acids, homology: 53%), while other chemokines from this group (IP-10 and MIG) are only 29% homologous. The observation that synthetic variants of PF4, but not IP-10 or MCP-1 modified by the insertion of an ELR motif, acquire the capacity to interact with chemokine receptors CXCR1 and CXCR2 (Clark-Lewis et al., 1993), highlights the close relationship of PF4 to chemokines of the ELR subfamily. There is no murine equivalent for the TG proteins. The only protein cloned from other species and identified as a homolog of PBP (61% sequence similarity) derives from porcine platelets (Power et al., 1995) (Figure 7). Although its N-terminus does not contain the cleavage site required for generation of a molecule corresponding to human NAP-2, N-terminally different variants with neutrophil-stimulating capacity have been shown to be released by porcine platelets (Yan et al., 1993).
Figure 6 Comparison of NAP-2 primary and secondary structure to other CXC chemokines. Sequence alignment of the human NAP-2, PF-4, MGSA, and IL-8 molecules. Amino acid residues identical in all four chemokines are boxed in green, and conservative exchanges are boxed in yellow. The location of helical (underlined) strand (double lines) secondary structural elements is indicated for PF4, NAP-2, MGSA, and IL-8 according to Zhang et al. (1994), Malkowski et al. (1995), Kim et al. (1994), and Clore et al. (1990), respectively. The number of residues identical to those in NAP-2 (id) is shown at the right. (Full colour figure can be viewed online.)
Figure 7 Sequence alignment of human and porcine PBP. A comparison of the cloned sequences of human (Wenger et al., 1989) and porcine PBP (Power et al., 1994) is shown. Including the leader sequences, the proteins are 61.0% homologous (identical amino acid residues are boxed in green). The cleavage sites correlating to the N-termini of truncated native isoforms are indicated by arrows. The porcine isoforms L-NAP-2 and S-NAP-2 do not correspond to human NAP-2 and are directly secreted by platelets (Yan et al., 1993). (Full colour figure can be viewed online.)
1084 Ernst Brandt, Andreas Ludwig and Hans-Dieter Flad
Posttranslational modifications Glycosylation The TG proteins do not contain consensus sequences for N-glycosylation, nor has O-glycosylation been reported. However, nonenzymatic glycosylation of lysyl residues in CTAP-III has been described (Castor et al., 1990). Only one type of monosaccharide, glucose at a moderate ratio (0±4 moles/mol of protein) was found, the number of sugar residues changing with the preparation analyzed. This type of glycosylation had no apparent influence on the biological activities of the chemokine. Deamination Further microheterogeneity in CTAP-III is caused by deamination of the N-terminal asparagine. This variant (CTAP-III/Asp1) is commonly present, along with authentic CTAP-III (CTAP-III/Asn1) in preparations of the chemokine from isolated platelets as well as from leukocyte culture supernates (Brandt et al., 1989; Van Damme et al., 1989; Castor et al., 1990). Deamination has no impact on CTAP-III biological activity. Limited Proteolysis The posttranslational modification most relevant to the biological activities of TG proteins consists of limited proteolysis of these molecules, at both their N- and C-termini. Apart from the major isoforms, PBP, CTAP-III, TG, and NAP-2, trace amounts of a variety of other N-terminally truncated variants have been found, both in unstimulated platelets and in platelet suspensions co-cultured with blood leukocytes. Unidentified platelet-derived as well as leukocyte-associated proteases are likely to be involved in the processing of the molecules. While N-terminal truncation does not appear to affect the connective tissue-activating properties of TG proteins (Castor et al., 1990), it clearly has an impact on their capacity to activate neutrophil functions. This is most strikingly illustrated by CTAP-III, the major TG protein stored and secreted by platelets, which by itself is inactive on neutrophils, but acquires high activity upon its cleavage into NAP-2 by monocytes (Walz and Baggiolini, 1990) and even more efficiently when cleaved by neutrophils themselves (Brandt et al., 1991; HaÈrter et al., 1994). In the case of neutrophils there is some evidence for plasma membraneassociated cathepsin G being the processing enzyme. While it has been observed that the neutrophil-stimulating capacity of TG proteins increases with the degree of N-terminal truncation, relevant activity
(i.e. at least 10% of NAP-2 potency) is only found in NAP-2 variants bearing N-terminal extensions of maximally three amino acid residues. C-terminal truncation was first observed in NAP-2 isolated from culture supernates of platelet-containing mononuclear blood cells (Brandt et al., 1993). Subsequently native variants of NAP-2 lacking four (NAP-2[1±66]) and seven amino acids (NAP-2[1±63]) at the C-terminus were isolated and identified (Ehlert et al., 1995, 1998). These variants had higher neutrophil-stimulating potency and greater receptor-binding affinity (by about 3-fold and 5-fold, respectively) than the full-size chemokine (NAP-2[1±70]). Analyses of recombinant C-terminally truncated variants of NAP-2 revealed that truncation by itself was not responsible for enhancement of neutrophil-directed biological activity, but that successive removal of the three negatively charged C-terminal amino acids correlated with a progressive increase in potencies. The abundance of a native CTAP-III molecule truncated by four residues suggests that truncated NAP-2 variants may derive from correspondingly truncated precursor molecules (Ehlert et al., 1998).
CELLULAR SOURCES AND TISSUE EXPRESSION
Cellular sources that produce See Table 3.
Eliciting and inhibitory stimuli, including exogenous and endogenous modulators The stimuli that induce PBP gene expression during megakaryocyte development have not been identified. Although it has been found that thrombopoietin, a major cytokine promoting megakaryocyte maturation, increases the synthesis of several platelet-specific -granule proteins including PF4 (Sasaki et al., 1997; Phillipp et al., 1998), a corresponding upregulation of PBP has not been reported. Interestingly, the platelet release products PBP/CTAP-III and PF4 themselves act back on megakaryocyte progenitors to inhibit their maturation (Han et al., 1990), thus indirectly decreasing platelet counts and consequently the availability of PBP/CTAP-III and PF4. Evidence has recently emerged that apart from megakaryocytes, other blood cells like neutrophils,
CTAP-III, TG, and NAP-2 1085 Table 3 Human cells and cell lines producing TG proteins Cell type
Remarks
Reference
Megakaryocytes
Synthesis during development
Holt et al., 1988
Platelets
Release from granules upon stimulation
Moore et al., 1975
Expression enhanced by DMSO or TPA Secreted in cell culture Enhanced by TPA or in contact with endothelial cells
Tabilio et al., 1984 Tanabe et al., 1991 Hoshi et al., 1994
Induced by PHA
Iida et al., 1996
Monocytes
Induced by LPS
Iida et al., 1996
Neutrophil granulocytes
Induced by LPS
Iida et al., 1996
Megakaryoblastic cell lines HEL MEG-01 CMK-7 T cells
monocytes, and T cells may be induced to produce and secrete so far unidentified variants of these chemokines (Iida et al., 1996). However, platelets remain the predominant source for secreted TG proteins (serum levels: up to about 30 mg/mL) with CTAP-III and PBP representing the major isoforms stored within these cells. While the relative amount of CTAP-III ranges from 60 to 90% (Brandt et al., 1991), PBP is present at a mean proportion of about 25%, with no TG detectable (Holt et al., 1988). Generally stimuli eliciting platelet aggregation and -granule release also induce the secretion of PBP/ CTAP-III. These include thrombin, the arachidonic acid metabolite thromboxane (TXA2), different types of conformationally intact collagen (e.g. types I, III, and VI which are found in the vessel wall), ADP, and PAF. Enhancement of platelet activation and granule release may also occur in response to neutrophil-derived cathepsin G (Chignard and Renesto, 1994). This protease is released following activation of neutrophils by stimulants such as fMLP and TNF . Cathepsin G activation of platelets is comparable to the potent agonist thrombin. Moreover, following its binding to platelets cathepsin G cleaves released PBP/CTAP-III into NAP-2 (Cohen et al., 1992). Prostaglandins PGE1 and PGI2 are major physiological inhibitors of secretion. The latter is released by endothelial cells upon stimulation by thrombin. Interestingly, TG has been reported to decrease endothelial PGI2 production, thus facilitating the formation of a locally restricted thrombus at sites of injury (Hope et al., 1979). Pharmacological inhibitors of secretion are phosphodiesterase inhibitors such as theophylline, activators of adenylyl cyclase such as hirudine, and synthetic ligands for prostacycline receptors, e.g. iloprost.
Because the formation of NAP-2, the major TG protein active on neutrophils, requires proteolytic cleavage of its platelet-secreted precursors PBP/ CTAP-III by leukocytes, the availability of NAP-2 is predominantly regulated at the processing level. From inhibition studies there is evidence that a cathepsin Glike serine protease which is constitutively expressed on the neutrophil plasma membrane (Brandt et al., 1991; HaÈrter et al., 1994), as well as so far undefined serine protease(s) and thiol protease(s) derived from monocytes (Walz and Baggiolini, 1990) are the major enzymes involved in processing. Accordingly, purified cathepsin G (Brandt et al., 1991; Car et al., 1991) as well as chymotrypsin (Car et al., 1991; Cohen et al., 1992), a protease exhibiting similar cleavage specificity, have been shown to generate active NAP-2 from its precursors. Major physiological inhibitors of NAP-2 generation reside in plasma and serum (HaÈrter et al., 1994), while leukocyte-derived elastase has the potential to degrade the chemokine (Car et al., 1991). Neutrophil-derived serine protease inhibitors such as aprotinin and chymostatin, as well as synthetic inhibitors like sulfonyl fluorides (PMSF, AEBSF), and chloromethyl ketones (Z-GLF-CK), block processing. Some of the monocyte-associated enzymatic activity is also susceptible to inhibition by leupeptin, a thiol protease inhibitor (Walz and Baggiolini, 1990).
RECEPTOR UTILIZATION See Table 4. Identification of NAP-2 Receptors Among TG proteins specific receptors have only been identified for NAP-2. Binding studies with the
1086 Ernst Brandt, Andreas Ludwig and Hans-Dieter Flad Table 4 Interaction of NAP-2 with chemokine receptors Receptor
CXCR1
CXCR2
DARC
Synonyms
IL-8RA/IL-8RI
IL-8RB/IL-8RII
Duffy
Affinity (Kd)
500 nM
1±4 nM
5 nM
5 nM
Expression
Neutrophils Monocytes Mast cells Basophils CD8+T cells NK cells
Neutrophils Monocytes Mast cells CD8+ T cells NK cells
Erythrocytes Endothelium
Herpesvirus saimiri
Signaling
Chemotaxis Degranulation [Ca2+]i flux PLD Respiratory burst
Chemotaxis Degranulation [Ca2+]i flux
None
[Ca2+]i flux
radiolabeled chemokines revealed that NAP-2, but not its precursors (PBP and CTAP-III) specifically interact with neutrophil receptors that also bind IL-8 and MGSA/GRO. More detailed comparison of the kinetics of competition for binding sites of these chemokines led to the prediction of the existences of two distinct populations of binding sites on neutrophils with characteristic differences in the binding capacities existing between NAP-2 and MGSA on the one hand and IL-8 on the other (Moser et al., 1991; Schumacher et al., 1992; Petersen et al., 1994). These binding sites were cloned and identified as the Gi protein-coupled heptahelical receptors CXCR1 and CXCR2 (previously termed IL-8RA and IL-8RB, respectively) (Holmes et al., 1991; Murphy and Tiffany, 1991). Following transfection into eukaryotic cell lines CXCR2 was shown to bind IL-8, NAP-2, and MGSA/GRO with high affinity (Kd=1±4 nM), whereas CXCR1 exhibited comparable high affinity for IL-8 only and considerably lower affinity for NAP-2 and MGSA (Kd=500 nM) (Loetscher et al., 1994; Ahuja and Murphy, 1996; Hoch et al., 1996; Murphy, 1997) (see Table 4). These receptors are coexpressed in high numbers predominantly on neutrophils, but are also detectable on monocytes, NK cells, CD8T cells, mast cells, and basophils by immunofluorescence labeling (Chuntharapai et al., 1994; Morohashi et al., 1995; Lippert et al., 1998; Ochsenberger et al., 1999). Several other structurally related receptors with high affinity for NAP-2 (and related chemokines) namely the Duffy antigenassociated receptor for chemokines (DARC) (Neote et al., 1993) on erythrocytes and endothelial cells, as well as herpesvirus-encoded CXC chemokine receptors (ECRF3) (Ahuja and Murphy, 1993), have been
ECRF3
identified. However, to date only CXCR1 and CXCR2 have been shown to mediate NAP-2-induced cellular responses such as chemotaxis (Loetscher et al., 1994; Ben-Baruch et al., 1997a). Structure±Function Relationship Like with IL-8 and MGSA/GRO, the N-terminal Glu-Leu-Arg- (ELR-) sequence motif of NAP-2 has been demonstrated to be an absolute requirement for specific binding to CXCR1 and CXCR2 (Yan et al., 1994; Clark-Lewis et al., 1994). In addition to the ELR motif, other regions on NAP-2 also contribute to receptor binding. The ultimate C-terminal amino acids which are not integrated into the helix and form fraying ends appear to disturb receptor binding, since their removal leads to the enhancement of binding affinity (Ehlert et al., 1995, 1998). However, C-terminal truncation including the leucine at position 63 decreases receptor binding, indicating that the L63, which is highly conserved in CXC chemokines, represents a functionally important residue. Several intersubtype receptor chimeras have been constructed to elucidate the structural basis for the differential affinity of CXCR1 and CXCR2 for NAP-2 and related chemokines. Switching of the N-terminal extracellular part between the receptor types demonstrated that the N-terminus of CXCR2 to a large extent dictates that NAP-2 (as well as MGSA/GRO) is bound with high affinity, while the analogous domain of CXCR1 does not appear to contain a high affinity binding domain(s) for these chemokines (LaRosa et al., 1992; Gayle et al., 1993). Detailed analyses of other receptor chimeras led to the discovery that additional regions on the second extracellular loop of
CTAP-III, TG, and NAP-2 1087 CXCR2 operate as high affinity determinants for NAP-2 (and MGSA/GRO), independently of the N-terminus (Ahuja et al., 1996). Receptor Regulation and Utilization by NAP-2 In accordance with its high and low affinity for CXCR2 and CXCR1, respectively, NAP-2 displays differential capacity to downregulate the two receptor types. Exposure to low concentrations of NAP-2 leads to the selective phosphorylation of CXCR2 at intracellular serine residues (Ben-Baruch et al., 1997b) and to the disappearance of the receptor from the cell surface (Ludwig et al., 1997). Neutrophils treated in such a way are functionally desensitized to subsequent stimulation with the same low dosage of NAP-2, but they are still responsive to elevated concentrations of the chemokine, presumably due to stimulation through CXCR1 (Ludwig et al., 1997). To achieve comparable downregulation of CXCR1 and a complete desensitization of NAP-2-induced responses much higher NAP-2 dosages are required. Although precursor CTAP-III neither binds to CXCR1 nor to CXCR2, it has the capacity to desensitize neutrophil responses, similar to NAP-2 (HaÈrter et al., 1994; Ehlert et al., 1998). This effect is due to the proteolytic conversion of CTAP-III into NAP-2 by neutrophils (see previous section), followed by receptor downregulation on the processing cells. Corresponding to its distinct binding affinities, NAP-2 exhibits differential capacity to activate either CXCR1 or CXCR2. This was shown by transfection of either receptor type into eukaryotic cell lines. While NAP-2 acts as a highly potent inducer of calcium flux and chemotaxis in CXCR2-transfected cells, it is considerably less potent in activating CXCR1-transfected cells (Loetscher et al., 1994; Ahuja and Murphy, 1996; Ben-Baruch et al., 1997a). The role of CXCR1 and CXCR2 in NAP-2-induced chemotaxis of neutrophils was elucidated by means of neutralizing anti-receptor monoclonal antibodies. These studies demonstrated that both receptors contribute to the migration of neutrophils along a concentration gradient of NAP-2, with CXCR2 rendering the cells responsive to low dosages of the chemokine, and with CXCR1 extending their responsiveness to much higher dosages (Ludwig et al., 1997). In contrast to these results, CXCR1 but not CXCR2 was found to mediate activation of PLD and NADPH-oxidase in neutrophils (Jones et al., 1996). This is consistent with the observation that NAP-2 functions as a chemotactic mediator rather than as an agonist for neutrophil respiratory burst (L'Heureux et al., 1995), while IL-8 does both. Thus due to their differential interaction with the two receptors, the related chemokines
NAP-2 and IL-8 induce distinct activity profiles in neutrophils.
IN VITRO ACTIVITIES
In vitro findings The TG proteins are active against a variety of target cells including endothelium and connective tissue as well as leukocytes and lymphocytes (Table 5). Although specific receptors for the NAP-2 precursors PBP, CTAP-III, and TG have not been identified, several cell types, such as megakaryocytes, fibroblasts, and endothelial cells respond to these molecules with distinct responses. Since these cell types are obviously not able to convert the precursors into NAP-2, and some of them do not even express chemokine receptors CXCR1 and CXCR2, it may be that ligand binding to surface proteoglycans or as yet unidentified receptors could be responsible for cell activation. Among the precursors, CTAP-III appears to function as a growth factor and as a mediator involved in tissue repair, based on its various stimulatory effects on connective tissue metabolism. However, CTAP-III may also have a role as a regulator of leukocyte functions, e.g. as a downmodulator of neutrophil activation and mediator of histamine release from basophils. Since several of the functions elicited by CTAP-III are also inducible by equimolar doses of NAP-2, N-terminal processing appears to be without importance. This is different from the neutrophildirected actions of TG proteins, where only NAP-2 and some minor variants being extended by up to three amino acids, have a potential for cell activation (Table 5). Similarly NAP-2 was reported to inhibit proliferation and maturation of megakaryocyte progenitors with 1000-fold greater potency than CTAPIII and to require the presence of the ELR motif, suggesting the involvement of CXCR2. Some unexpected reactivity profiles have been found for the less well-defined TG protein variants. While LPS-stimulated monocytes were reported to produce an N-terminally extended isoform of PBP that induced fibroblast proliferation with a potency comparable to that of PDGF, a native NAP-2 variant was found to exhibit heparinase-like enzymatic activity.
Regulatory molecules: Inhibitors and enhancers The functional activity of NAP-2 for neutrophils (degranulation, chemotaxis) is inhibited following
1088 Ernst Brandt, Andreas Ludwig and Hans-Dieter Flad Table 5 CTAP-III/ TG/NAP-2 in vitro biological activities Target cell
TG protein
Function
Reference
Megakaryocytes
TG, CTAP-III
# Megakaryocytopoiesis
Han et al., 1990
NAP-2
# Megakaryocytopoiesis
Gewirtz et al., 1995
NAP-2
" Survival
Han et al., 1997
Hematopoetic progenitor cells
# Chemosensitivity Fibroblasts
TG
" Chemotaxis
Senior et al., 1983
CTAP-III, NAP-2
" Synthesis of matrix components
Castor et al., 1990
CTAP-III, NAP-2
" Glucose transport and GLUT-1 glucose transporter expression
Tai et al., 1992
PBP-precursor
" Proliferation
Iida et al., 1996
Endothelial cells
TG
# PGI2 production
Hope et al., 1979
Neutrophils
CTAP-III/NAP-2
# NAP-2/IL-8-induced degranulation
HaÈrter et al., 1994; Petersen et al., 1994
NAP-2
" [Ca2+]i flux
Walz et al., 1991
NAP-2
" Chemotaxis
Walz et al., 1989
NAP-2
" Degranulation
Walz and Baggiolini, 1989
NAP-2
" Adhesion receptors
Detmers et al., 1991
NAP-2
" Respiratory burst (weak)
Walz et al., 1991
NAP-2
" Fc receptor-dependent chemiluminescence
Brandt et al., 1989
NAP-2
" Phagocytosis of iGg-coated erythrocytes
Detmers et al., 1991
NAP-2
" [Ca2+]i flux
Krieger et al., 1992
CTAP-III, NAP-2
" Histamine release
Kuna et al., 1993
Basophils
2+
Mast cells
NAP-2
" [Ca ]i flux " Chemotaxis
Lippert et al., 1998
Eosinophils
NAP-2
" [Ca2+]i flux
Schweizer et al., 1994
Mononuclear cells
NAP-2
" NK and LAK activity
Klein-Struckmeier et al., 1991
Lymphocytes
NAP-2
# IL-2-induced tumor cytolytic activity
Klein-Struckmeier et al., 1991
Other
NAP-2 variant
" Heparinase enzymatic activity
Hoogewerf et al., 1995
preincubation of the cells with CTAP-III. The mechanism may be termed homologous desensitization, because the NAP-2 generated during processing of CTAP-III by neutrophils downmodulates receptors for NAP-2 (HaÈrter et al., 1994). Preincubation of neutrophils with TNF primes the degranulation response to NAP-2 (Brandt et al., 1992). In basophils NAP-2-induced histamine release is enhanced by priming with IL-3 (Krieger et al., 1992).
Bioassays used See Table 6.
Lippert et al., 1998
IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS
Normal physiological roles In vivo activities of human NAP-2 were tested along with those of IL-8 and GRO in experimental rat models by both intradermal and intravenous injection (Zwahlen et al., 1993). Intradermal administration of human IL-8 induced rapid and concentrationdependent neutrophil infiltration which peaked 4
CTAP-III, TG, and NAP-2 1089 Table 6 Bioassays used for the determination of TG protein activities Target cell
Function
Assay
Reference
Megakaryocytes
Megakaryocytopoiesis
Colony formation in plasma clots
Han et al., 1990; Gewirtz et al., 1995
Hematopoietic progenitor cells
Survival, chemosensitivity
Colony formation in plasma clots
Han et al., 1997
Fibroblasts
Chemotaxis
Blind well Boyden chamber assay on micropore membranes
Senior et al., 1983
Synthesis of matrix components
[14C]Glucosamine incorporation into [14C]glycosaminoglycan
Castor et al., 1990
Glucose transport
2-deoxy[14C]glucose uptake
Tai et al., 1992
3
Proliferation
[ H]Thymidine incorporation
Iida et al., 1996
Endothelial cells
PGI2 production
Inhibition of arachidonic acid-induced platelet aggregation
Hope et al., 1979
Neutrophils
[Ca2+]i flux
Fluorescence of Fura-2-loaded cells
Walz et al., 1991
Chemotaxis
Blind well Boyden chamber assay on micropore membranes
Walz et al., 1989
Respiratory burst
Luminol-dependent chemiluminescence
Walz et al., 1991
Fc receptor-dependent chemiluminescence
Luminol-dependent chemiluminescence in the presence of IgG-coated erythrocytes
Brandt et al., 1989
Phagocytosis of IgG-coated erythrocytes
Counting of erythrocytes remaining neutrophil-associated after washing
Detmers et al., 1991
Degranulation
Release of elastase enzymatic activity by cytochalasin B-treated cells
Walz and Baggiolini, 1989
Basophils
Histamine release
Mononuclear cells
NK and LAK activity
Kuna et al., 1993 Release of radioactivity from Eu3+/diethylenetriaminepentaacetic acid-loaded K562 and Raji cells, respectively
hours after application. Injection of GRO induced similar chemotactic responses, whereas NAP-2 was significantly less active. Similar observations were made in rabbits, where intradermal injection of NAP2 and IL-8 revealed IL-8 to be considerably more potent in the induction of neutrophil accumulation and plasma leakage than NAP-2 (Van Damme et al., 1990). The role of kidneys in the metabolic clearance of TG proteins and PF4 was examined by injecting supernates of aggregates of thrombin-stimulated platelets into sham-operated control rats, nephrectomized rats, and into rats with acute ureteral ligation (Bastl et al., 1981). The half-lives for the fast and slow
Klein-Struckmeier et al., 1991
components of TG antigen in control rats were 6.4 and 68.4 minutes, respectively. Nephrectomy significantly increased the time to 9.7 and 144 minutes, while ureteral ligation resulted in no significant change. Less than 0.1% of the total TG antigen injected was recovered in the urine of control rats. In contrast, the clearance of PF4 was not affected by nephrectomy. Taken together, the findings indicate that functional renal tissue is necessary for normal clearance of TG proteins, but renal excretion does not play a major role in its elimination, suggesting that the protein is catabolized in the kidney. Catabolic clearance of PF4 does not depend on functioning kidney tissue.
1090 Ernst Brandt, Andreas Ludwig and Hans-Dieter Flad
PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY
Normal levels and effects The physiological concentrations of TG proteins in human plasma have been determined to range around 6 ng/mL, while concentrations in serum (about 14 mg/ mL) are > 1000-fold higher (Files et al., 1981). At plasma levels, neither CTAP-III nor NAP-2 can be envisaged to exhibit any of their cell-directed effects, as suggested by the much higher concentrations needed to elicit functions in vitro. Thus, platelet activation is obviously required to provide functionally relevant levels of these chemokines.
Role in experiments of nature and disease states Inflammatory Reactions TG proteins, PF4, and RANTES are released from platelets during storage and accumulate over time in platelet concentrates (Bubel et al., 1996). These chemokines may play a causative role in nonhemolytic transfusion reactions because of their inflammatory potential. Gingival tissue inflammation is also associated with platelet activation, since TG was detected in gingival tissue inflammation at concentrations of 5±45 ng/mL (Steinberg et al., 1995). Rheumatoid Arthritis, Osteoarthritis To gain insight into the pathophysiological role of TG proteins, CTAP-III ( TG/NAP-2) isoforms were measured in platelets and in plasma of normal persons and those with rheumatoid arthritis (Castor et al., 1993). CTAP-III was the primary isoform of this antigen family in normal platelets and in plasma, TG and NAP-2 accounted for systemic sclerosis, and 15% of those with systemic lupus erythematosus (Myers et al., 1980). The CTAP-III content in platelets of patients with rheumatoid arthritis was found to be decreased compared with normal subjects (Smith and Castor, 1978). Normal human spleen and kidney contained substantial (mg/gm) amounts of CTAP-III and traces of NAP-2, whereas the concentrations of CTAP-III in liver, lung, and urine were lower (ng/ gm). This suggests that deposition of CTAP-III in tissues is associated with partial processing to NAP-2 isoforms with the potential to induce activation of
neutrophils and fibroblasts in patients with a rheumatic disease. CTAP-III isoforms, including NAP-2, were detected in synovial tissue from patients with rheumatoid arthritis and osteoarthritis (Castor et al., 1992). The biological activity was determined by measuring the uptake of [14C]2-deoxyglucose into glycosaminoglycan and [3H]TDR uptake into DNA of synovial cell cultures. Both CTAP-III and NAP-2 synergized with recombinant IL-1 , basic fibroblast growth factor, and prostaglandin E1 and E2, whereas recombinant PDGF-BB and recombinant TGF exhibited additive effects on glycosaminoglycan synthesis. The results suggest a regulatory role of CTAPIII and its isoforms in combination with other endogenous cytokines on synovial cell metabolism in rheumatoid arthritis and osteoarthritis. Wegener's Granulomatosis, Scleroderma, Progressive Systemic Sclerosis Activation of neutrophils is a hallmark of Wegener's granulomatosis, a special form of vasculitis. Preliminary data of our group have shown that the CTAP-III processing capacity of neutrophils is impaired compared with normal controls (Trabandt and Brandt: unpublished observations). In patients with scleroderma and progressive systemic sclerosis elevated levels of hyaluronate in the serum reflect enhanced synthesis or outflow of hyaluronate from the connective tissue (Engstrom-Laurent et al., 1985). Interestingly, in a subgroup of patients elevated plasma TG levels were measured and found to correlate with circulating platelet count and with the highest hyaluronate levels. PDGF, also stored in granules of platelets, like TG, stimulates hyaluronate synthesis of fibroblasts, indicating a further fibroblast-stimulating effect of proteins released by platelets in scleroderma. Renal Diseases Patients with end-stage kidney disease were reported to have marked elevations of plasma CTAP-III levels (Castor et al., 1993). In addition to fibrinogen derivatives such as fibrinopeptide A (PFA) and beta 15-42 antigen fragment E, elevated plasma levels of TG were described (Lane et al., 1984). This condition presumably reflects an impairment of TG elimination rather than increased production. Adult Respiratory Distress Syndrome and Congestive Heart Failure The levels of NAP-2 were studied in patients with adult respiratory distress syndrome (ARDS) or congestive
CTAP-III, TG, and NAP-2 1091 heart failure (CHF) with pulmonary edema (Cohen et al., 1993). Pulmonary edema fluids from patients with CHF contained higher concentrations of TG antigen than pulmonary edema fluids (PEFs) from patients with ARDS, who in turn had elevated levels in their bronchoalveolar lavage (BAL) fluid as compared with BAL fluid from normal control subjects. The TG antigen concentrations were 4.1-fold greater in pulmonary edema fluids from patients with congestive heart failure than in their plasmas, and the PEFs contained chemotactic activity, indicating the presence of NAP-2. These data suggest significant degranulation of platelets in the lungs of patients with chronic heart failure. Furthermore, in patients with chronic congestive heart failure the elevated levels of TG and PF4 can be decreased by treatment with dipyridamole (Zhang and Ling, 1995). Elevated plasma levels of TG and PF4 were also described in patients with depression and ischemic heart disease (Laghrissi-Thode et al., 1997). Thus NAP-2 generated by proteolytic processing of TG/CTAP-III may recruit neutrophils to the site of an atheromatous lesion. Coronary Artery Disease Platelet activation also occurs in coronary artery disease, since platelets are the main components of arterial thrombi, and since they also contribute to the development of atheroma and smooth muscle hypertrophy in arterial walls (Cahill and Newland, 1993; Ulutin et al., 1997). Sickle Cell Anemia In steady-state sickle cell anemia TG and PF4 levels are significantly higher than in controls, but the TG : PF4 ratio is lower in patients than in controls (Adamides et al., 1990). This finding in the absence of any statistical correlation between platelet number and TG or PF4 indicates that platelets seem to be activated in sickle cell syndromes both in homozygous and sickle cell/ thalassemia heterozygotes. Heparin-Associated Thrombocytopenia It is of interest to note that 6 out of 15 patients with heparin-associated thrombocytopenia had autoantibodies to IL-8, and 3 out of 15 had autoantibodies to NAP-2 (Amiral et al., 1996). Plasma TG determinations can be useful in determining the mechanisms of thrombocytopenia of various etiologies, since TG is considered to be a marker of intravascular degranulation (Rubenstein and Wall, 1981).
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