Platelet Adhesion Receptors

PLATELET A DHESI0N RECEPTORS Kenneth J . Clemetson I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 I1. Platelet Glycoproteins with a Role in Adhesion . . . . . . . . . . . . . . . . . . 33 111. Structure of the Glycoprotein Ib-V-IX Complex . . . . . . . . . . . . . . . . . 35 A . Glycoprotein Iba . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 38 B . Glycoprotein IbP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. GlycoproteinIX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 D. GlycoproteinV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 40 E . Polymorphism within GPIba . . . . . . . . . . . . . . . . . . . . . . . . . IV. Function of the GPIb-V-IX Complex . . . . . . . . . . . . . . . . . . . . . . . 40 A. Bleeding Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 43 B . Biochemical Evidence for Function . . . . . . . . . . . . . . . . . . . . . C. The Binding Site for GPIb on vWf . . . . . . . . . . . . . . . . . . . . . . 44 D. Non-physiological Activators of the GPIbhWf Axis . . . . . . . . . . . .45 E . The Role of the GPIb-V-IX Complex in Thrombin Activation of Platelets . 47 F. Expression of GPIb-V-IX . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 G. Platelet Activation and Signal Transduction Via the GPIb/vWf Axis . . . . 49 V. Other Adhesion Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 49 A . Collagen Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 B . Fibronectin and Laminin Receptors . . . . . . . . . . . . . . . . . . . . . C. Thrombospondin and Its Receptors in Adhesion . . . . . . . . . . . . . . . 5 1 D. The Vitronectin Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 E . GPIIb-IIIa in Adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Advances in Molecular and Cell Biology. Volume 18. pages 31-66 Copyright 0 1997 by JAI Press Inc All rights of reproduction in any form reserved ISBN: 0-7623-0140-6

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KENNETH J. CLEMETSON

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VI. Inhibition of Platelet Adhesion as a Prophylactic Measure

or for Treatment of Acute Thrombotic Events . . . . . . . . . . . . . . . . . . 53 54 A. P-Selectin (CD62, GMP-140, PADGEM) . . . . . . . . . . . . . . . . . 54 B. PECAM-1 (Platelet and Endothelial Cell Adhesion Molecule, CD31) . . . 56

VII. Adhesion of Platelets to Other Cells . . . . . . . . . . . . . . . . . . . . . . .

1. INTRODUCTION Platelets have an essential role in hemostasis, the prevention of bleeding from damaged blood vessels, as can easily be seen from the problems arising in severe thrombocytopenia. Although they contain many components that are important for these h c t i o n s , surface glycoproteins are critical for two processes, adhesion and aggregation. In platelets, adhesion has a restricted definition, referring to the attachment of platelets to subendothelium or to other cells, while platelet-platelet “adhesion” is referred to as aggregation to differentiate these processes clearly (Figures 1A and 1B). Primary adhesion is the binding of resting platelets to subendothelium (Figure 1A) and secondary adhesion the binding of activated (via unsatisfactory primary adhesion or temporary association with a thrombus) to subendothelium (Figure 1B). The adhesion mechanisms are therefore not completely identical. When vessel wall is damaged and endothelial cells removed or cell-cell junctions interrupted exposing the extracellular matrix of the suben-

0 UNACTIVATED PLATELETS

OM

0 A -ARE ACTIVATED

ASPREAD

ENDOTHELIAL CELLS

vWF DEPOSITED ON SUBENDOTHELIUM (continued

Figure 7. Schematic drawing of the processes involved in: (a) primary platelet adhesion (b) Platelet aggregation and secondary adhesion. Note that activated platelets adhering downstream can come from unsuccessful upstream interactions with either subendothelium or a thrombus.

platelet Adhesion Receptors

33

0 UNACTIVATED PLATELETS

OA

d' CONTA

0 AGGRE 0

ENDOTHELIAL CELLS

vWF DEPOSITED ON SUBENDOTHELIUM Figure 1. (Continued)

dothelium, platelets adhere, are activated, spread, release storage granule contents and bind further platelets and, eventually, also some leukocytes and monocytes. The molecular steps involved in these processes are at least partly, if not completely, understood. Probably, different receptors are involved to different extents depending on the local conditions but we can now propose a model for the overall process that fits most of the observed facts. In this chapter the platelet surface receptors involved in adhesion will be described and their structure/function relationships is implicated particularly in aggregation and discussed. Since GPIIb-IIIa (aIIb&) will be dealt with in detail in Chapter 3 by Drs. Abrams and Shattil it will be covered only briefly here in connection with its role in spreading. Similarly, the collagen receptor(s) will be dealt with in detail in Chapter 4 by Drs. Santoro, Saelman, and Zutter and the reader is referred to this to supplement the coverage given here.

II. PLATELET GLYCOPROTEINS WITH A ROLE IN ADHESION Platelets contain a wide variety of membrane glycoproteins many of which are critical for adhesion or aggregation (see Figure 2). When platelets come into contact with exposed subendothelium the receptors participating in interactions seem to depend upon the shear stress to which the platelets are exposed. Evidence for this comes from perfusion chamber studies done with reconstituted blood and also with platelets lacking specific glycoproteins or by using specific antibodies to block function (Weiss et al., 1978, 1986; Sakariassen et al., 1987; Coller et al., 1983). These studies have pointed to the importance of the von Willebrand factor (vWf)/GPIb axis as critical for platelet adhesion at high shear as found in

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KENNETH J.CLEMETSON

Figure2. Fluorogramof a two-dimensional isoelectricfocusing/SDS-polyacrylamide gel of resting platelets, surface-labeled using periodate/[3H]-NaBH4 to show the principal glycoproteins involved as adhesion receptors. P-Selectin is only seen when activated platelets are labeled.

capillaries or in larger vessels under atherosclerotic conditions but it is probably also important at low shear. Since, however, even BernardSoulier syndrome (BSS) patients, completely lacking GPIb-V-IX, normally only have bleeding problems after considerable trauma there must exist other, parallel, mechanisms capable of substituting for the vWf7GPIb axis to a considerable extent and which fail to

Platelet Adhesion Receptors

35

compensate fully only under very rigorous circumstances. It is the aim of this review to consider the various glycoproteins involved in the adhesion process and how they cooperate in order to fblfill the functions required for normal primary hemostasis. Thus, most of the evidence that we have available points to the paramount importanceof GPIb/vWf in this process since the lack of either of these components produces the most severe consequences either in vivo or in in vitro perfusion chamber experiments (Weiss et al., 1978, 1986; Sakariassen et al., 1987).However, it is also clear that GPIb/vWfdoes not act alone and that other platelet glycoproteins are known to take part in the succeeding stages leading to the rapid spreading of platelets to form a protective and repair-inducing layer over an injury site. Since these same processes in an exaggerated form may lead to pathological situations it is important to understand how they are regulated and controlled and how the various pathways communicate to maintain this delicate balance.

111. STRUCTURE OF THE CLYCOPROTEIN Ib-V-IX COMPLEX The glycoprotein (GP) Ib-V-IX (CD49a, b, c, and d) complex consists offour chains each coded by separate genes present on different chromosomes. GPIb (CD49b and c) contains GPIba (150 kDa; CD49b, gene on chromosome 17p12-ter; Wenger et al., 1989) and GPIbP (27 kDa; CD49c, chromosome 22; Bennett, 1990) linked by a disulfide bond (Phillips and Poh Agin, 1977) while GPIX (CD49a, 22 kDa, gene on chromosome 3, Hickey et al., 1990) is strongly non-covalently associated in a 1:l ratio (Du et al., 1987) and GPV (CD49d, 82 kDa) weakly non-covalently associated with the complex in a 1:2 ratio (GPV:GPIb) (Modderman et al., 1992; see Figure 3). There are around 25,000 copies of GPIb-IX per platelet (Bemdt et al., 1985). A. Glycoprotein Iba

GPIba consists of several distinct domains (Lopez et al., 1987) which have different roles in its overall function. The N-terminal region, which was also directly sequenced as protein (Titani et al., 1987), consists of a loop formed by a disulfide bond followed by a leucine-rich domain consisting of six and one-half repeats of a 24 amino acid, leucine-rich sequence very similar to that found in an increasing number of proteins (for a review see Roth, 1991). All of the GPIb complex proteins contain this motif. The first such protein to be described was the leucine-rich glycoprotein found in plasma, containing nine such domains (Takahashi et al., 1985), the function of which is still unknown. These domains form P-pleated sheets a-helix loops (Gay et al., 1991; Krantz et al., 1991) and associate to form wedge-shaped, arc- or horseshoe-like structures. Table 1 lists some typical proteins containing these repeats. This domain also contains a single free cysteine

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KENNETH J.CLEMETSON

Actin Filaments Figure 3. Schematic drawing of the GPlb-V-IX complex, indicating the major domains of the four subunits, known binding sites, phosphorylation and acylation sites, and known interactions with the cytoskeleton.

residue. The apparent low reactivity of the thiol group may be due to it being buried in the middle of the leucine-rich sequences but there is recently some evidence for a population of dimers linked by this residue (Clemetson and Hiigli, 1994). Following this region two disulfide bonds form an overlapping double loop (Hess et al., 1991). This domain is important for the binding sites of the molecule (see Figure 4) and will be dealt with in more detail later. Just below the double loop comes a sequence rich in negatively charged amino acids that then switches abruptly to positively charged and is then followed by a domain with five, nine amino acid, repeats rich in threonine, and serine residues that are 0-glycosylated. The structureof this region stronglyresemblesthat ofthe mucins with a high density of short 0-linked oligosaccharides. Before reaching the membrane there is an unglycosylated region andjust above the membrane is the cysteine forming the link to GPIbP. In fact there are two cysteines here and it is still not known which of

Platelet Adhesion Receptors

37

Table 7. Some Proteins Containing Leucine-rich Repeats' Protein

Species

Repeats

Consensus Sequence

Reference

Leucine-rich a2-glycoprotein Platelet CPlba Platelet CPlbp Platelet GPlX Platelet GPV

Human

8

PPCLLQCLPQLR-LDLSCN-LESL Takahashi et al. (1985)

Human Human Human Human

7

15

P-CLL-LP-L--L-LS-N-LTTL P-CLL--LP-L--L-LS-N-LTTL P-CLL--LP-L--L-LS-N-LTTL P--LF--L--L--L-L--N-L--L

Toll

Drosophila

15

Chaoptin Slit RNAse inhibitor Carboxypeptidase N

Drosophila Drosophila Human Human

41 22 7 12

Note:

1

1

L6pez et al. (1987) L6pez et al. (1988) Hickey et al. (1989) Hickey et al. (1993) Lanza et al. (1993) Hashirnoto et al. (1988) Reinke et al. (1988) Rothberg et al. (1990) Schneider et al. (1988) Tan et al. (1990)

' A large number of proteins containing leucine-rich domains are now known. Those shown here have the highest similarity to the CPlb complex. In addition, there are flanking regions to many of these domains which also show high degrees of similarity.

Elastase

v

EEDTEGDKVRATRTWKFP

A

290

Figure 4. Detailed diagram of the double-loop region of GPlba, showing cathepsin G and elastase primary cleavage sites and sequences implicated in thrombin and von Willebrand factor binding based on studies with peptides and antibodies. The sequence of the 40 amino acid loop from Phe216-Thr240 has been implicated as a thrombin binding site while the region from Asp235-Lys262 has been implicated in von Willebrand factor binding. The segment from Asp269-Asp287 is highly charged and shows some similarity to the C-terminal peptide of hirudin involved in binding to the exosite of thrombin.

KENNETH J. CLEMETSON

38

these forms the link. However, it seems unlikely that both are involved because of the cysteine distribution on GPIbP (it would then also have an uneven number) and it seems more likely that the lower cysteine is imbedded in the lipid ofthe membrane at the start of the a-helix traversing the membrane and thus protected. The transmembrane region (29 amino acids) is followed by a 96 amino acid cytoplasmic domain that can associate with actin-binding protein (Andrews and Fox, 1991)and hence with the membrane-associated cytoskeleton. GPIba glycosylation has been intensively investigated. There are four putative N-glycosylation sites and biantennary, triantennary (Tsuji and Osawa, 1987) and tetraantennary monofucosylated chains (Korrel et al., 1988) have been described. There are many 0-glycosylation sites and the bulk of these are occupied by a biantennary hexasaccharide structure (Korrelet al., 1984;Tsujietal., 1983).Minoramountsofpenta-(Korreletal.,1985), tetra-, and tri-0-linked saccharideswere also detected by these authors. B. Glycoprotein Ibp

Although much smaller than the a-chain, the P-chain has certain similarities to

it (Lopez et al., 1988). The N-terminal region contains two disulfide loops followed

by a single 24 amino acid leucine-rich repeat then comes a further two disulfide loops, the single cysteine just above the membrane forming the link to the a-chain, the transmembrane region and a 34 amino acid cytoplasmicdomain. Just below the membrane lies a cysteine that can be palmitylated (Muszbek and Laposata, 1989) and, in the middle of the cytoplasmic domain lies serine 166 that can be phosphorylated (Wyler et al., 1986) by CAMP-dependentkinase (Wardell et al., 1989). It is not yet clear whether this domain is also involved in the association with actinbinding protein which can also be phosphorylated by CAMPdependent kinase (Cox et al., 1984). GPIbP has a single N-glycosylation site with a lactosamine biantennary oligosaccharidewithin the leucine-rich domain (Wicki and Clemetson, 1987). There is no 0-glycosylation. C. Glycoprotein IX

The structure of GPIX, overall, closely resembles that of GPIbP (Hickey et al., 1989, 1990). The N-terminal region contains two disulfide loops followed by a single 24 amino acid leucine-rich repeat then comes a further two disulfide loops, a short sequence before the transmembrane region, and a six amino acid cytoplasmic domain. Just within the membrane from the cytoplasmatic surface lies a cysteine that can be palmitylated (Muszbek and Laposata, 1989). As GPIbP it has a single N-glycosylation site with a lactosaminebiantennary oligosaccharide within the leucine-rich domain (Wicki and Clemetson, 1987). There is also no O-glycosylation.

39

platelet Adhesion Receptors

D. Glycoprotein V

GPV has recently been cloned by two groups (Hickey et al., 1993; Lama et al., 1993) so that the complete primary structure is now known (see Figure 5). A considerable part of the sequence had already been obtained by peptide sequencing (Shimomura et al., 1990). It shares many general structural features with the other members of the complex, in particular GPIba. Thus, from the N-terminus, it also contains two disulfide loops followed by 15 leucine-rich repeats, then two disulfide loops followed by the thrombin cleavage site (but no hirudin-like anionic site). After comes a sequence containing one N-glycosylation site and two 0-glycosylation sites but no mucin-like repeats. Then comes the transmembrane region and a 16 amino acid cytoplasmicdomain with no phosphorylation sites (no serine, threonine, or tyrosine residues). Overall there are eight N-glycosylation sites, with six of these in the leucine-rich repeat region. There are no palmitylation sites, supporting an

N-glycosylation

b

I I No phosphorylationsites in cytoplasmic domain

Figure 5. Schematic drawing of GPV showing putative disulfide loops and glycosylation sites and the sites of cleavage by thrombin and calpain.

KENNETH J.CLEMETSON

40

earlier conclusion based on labeling studies that it is not palmitylated (Muszbek and Laposata, 1989). The thrombin cleavage site does not appear to have a direct or indirect role in platelet activation by thrombin (Bienz et al., 1986), unlike the binding site on GPIba. A possible role in modulating GPIb-V-IX function was suggested by the inhibitory effect of alloantibodies from a Bernard-Soulier syndrome patient on ristocetin-induced aggregation of normal platelets (Drouin et al., 1989).

E. Polymorphism within GPlba No polymorphisms have yet been reported for the other members of the complex but several are known within GPIba. One ofthese, the Siba-Sibb,KO,HPA-2 system (Ishida et al., 1991; Kuijpers et al., 1992) responsible for alloantibody induction, is a Thr145 (89%)/Met145 (11%) (Murata et al., 1992) polymorphism. Another, seemingly with no immunological consequences, involves the duplication or tripling of a 13 amino acid sequence between Ser399 and Thr411 (Lopez et al., 1992). Originally, these size polymorphisms of GPIba were found in the Japanese population and described as A, B, C, and D from the highest to the lowest mass, with about 2,000 Da between each (Moroi et al., 1984). They were later reported from other populations as well (Jung et al., 1986). From molecular biology studies D was shown to be the molecule with the single sequence from Ser399 to Thr4 11, while C was the duplicate and B the triplicate form. The A form, which is much rarer in Caucasian populations, has recently identified as a quadruple form of this 13 amino acid sequence (Ishida et al., 1995). The size differences found can be explained on the basis of the 13 amino acid segment because it contains five threonine and serine residues that can be 0-glycosylated (see above). Since each hexasaccharide has a mass of about 1,200Da, the observed mass difference of more than 2,000 Da per additional segment would fit an average glycosylation on two sites.

IV. FUNCTION OF THE CPIb-V-IX COMPLEX A. Bleeding Disorders

Much of what we know about the function of the GPIb-V-IX complex comes from studies of inherited bleeding disorders where expression of this complex on the platelets is defective. Bernard-Soulier Syndrome

In the Bernard-Soulier syndrome (BSS) GPIb-V-IX is either absent, severely depleted or non-functional (George et al., 1984; Clemetson and Liischer, 1988). Indeed, it was studies of this disorder that provided most of the first evidence for the role of the GPIb complex (Nurden and Caen, 1975; Jenkins et al., 1976) and

Platelet Adhesion Receptors

41

for the association of GPIb with GPIX and GPV (Clemetson et al., 1982; Berndt et al., 1983). Similarly, such studies provided the first clues about the physiological role of this complex. A particularly important observation was that BSS platelets adhere poorly, if at all, to subendothelium at all shear rates (Weiss et al., 1974,1978) emphasizing the importance of the GPIb-V-IX complex in this primary phase of hemostasis. BSS is a rare, autosomal, recessive, genetic disorder.The long bleeding time, thrombocytopenia and morphologically abnormal, unusually large (“giant”) platelets are characteristic. Resting BSS platelets are incapable of interacting with vWf (Bithell et al., 1972; Howard et al., 1973) and therefore show dramatically decreased adhesion to subendothelium of damaged vascular wall. Aggregation to other agonists except thrombin (see below) is normal (Bithell et al., 1972). In the classic form of BSS all four chains are virtually completely absent (i.e., less than 1% can be detected) and the platelets are giant (up to the size of leukocytes) and have a more fluid membrane than normal. In virro they do not aggregate to either ristocetin or botrocetin in the presence of vWf (Zucker et al., 1977), nor directly to asialo vWf (De Marco and Shapiro, 198 1) or animal vWf such as bovine orporcine. Another characteristic difference between BSS and normal platelets is their reduced response to and binding of, thrombin (Jamieson and Okumura, 1978; Takamatsu et al., 1986). The thrombin receptor on BSS platelets is most probably normal and it is the absence of GPIb which causes this effect. GPIba contains a thrombin-binding site (Okumura et al., 1978; Harmon and Jamieson, 1986) which, when blocked by antibodies (Jenkins et al., 1983; Mazurov et al., 1991) or removed by proteolytic cleavage (Tam et al., 1980) reduces the response of normal platelets to a-thrombin. The cleaved form, y-thrombin, activates both normal and BSS platelets with similar kinetics but does not bind to GPIb (Jandrot-Permset al., 1988, 1990). BSS platelets also show differences in coagulant activity from normal (Walsh et al., 1975). Prothrombin consumption was reported lower than normal whereas platelet factor 3 (= surface exposure of negatively charged lipids) activity was raised (Perret et al., 1983). This latter may be due either to the increased size ofthe platelets, although other giant platelet syndromesdo not show the same effect, or to changes in the distribution of the lipid bilayer (Bevers et al., 1986). BSS platelets were also shown to have more easily deformable membranes than normal (White et al., 1984). Both these phenomena may also be associated with the absence of the GPIb-V-IX complex which, in resting platelets, is linked to actin-binding protein (Okita et al., 1985) and hence to the membrane associated cytoskeleton (Solum and Olsen, 1984; Fox, 1985). Over the years a number of variant BSS cases have been described (De Marco et al., 1990; Drouin et al., 1988; Poulsen and Taaning, 1990; Ware et al., 1991; Zwierzina et al., 1983). Although these patients show symptoms similar to those of the classic cases they show a wide variety of molecular differences. Thus, patients have been described with relatively normal levels of all four chains where the problem could eventually be localized to a point mutation in the outer domain of GPIba (Ware et al., 1991). In other cases the amounts of all the chains are reduced

KENNETH J. CLEMETSON

42

Mutation sites

GPlbu Leu-rich domains

Ser/Thr rich domains

E3TK2IIJ-L-! 1EC.- E I 3 C - m P e

GPIX

1

x 1 5 6 putative Several sites Trpj43 nonsense

Leu-rich domain

A A

*

Asp21 Asn45

v

Giy

Sbr

Figure 6. Diagram showing the location of the mutation sites in CPlba and GPIX known to cause Bernard-Souliersyndrome.

in parallel (Bemdt et al., 1983; Miller et al., 1992) suggesting that there is either a coordinated expression or that as with GPIIb/IIIa the expression level of one of the chains regulates the amount of the mature complex and excess amounts of the other chains are eliminated by degradation. Finally, there are a few rare cases where there is an imbalance between the chains expressed. Thus, low amounts of GPIX have been found in one case where GPIb was apparently totally absent (Hourdille et al., 1990) and we have seen cases where, at low levels of all the chains, there was clearly less GPIX than the other three (Drouin et al., 1988; Clemetson and Clemetson, 1994).As might be expected there are also rare cases which seem to be due to double heterozygote defects within the GPIba chain (Ware et al., 1990) and, more unexpectedly, a recent report of cases produced by double heterozygote point mutations in the gene for GPIX (Wright et al., 1993). Figure 6 shows the localization of the mutations in GPIba and GPIX so far identified in BSS. Platelet-type von Willebrand’s Disease

Other evidence for the role of GPIb-V-IX and its functions comes from a further bleeding disorder, platelet-type, or pseudo, von Willebrand’s disease where the platelets show an abnormal affinity for normal von Willebrand factor and as a result are aggregated and removed from the circulation (Miller et al., 1983). Such patients therefore develop thrombocytopenia and consequently bleeding problems. The molecular defect in platelet-type von Willebrand disease has been established in two families. In one family there is a G to T point mutation in the gene for GPIba causing a valine for glycine substitution at position 233 of the protein sequence (Miller et al., 1991). This substitution may lead to a different local peptide conformation and hence to the spontaneous binding of vWf to GPIb characteristic

platelet Adhesion Receptors

43

figure 7. Detailed diagram of the double-loop region of GPlba, showing the mutations that have been shown to cause “platelet-type” von Willebrand’s disease by increasing the binding of normal von Wiilebrand factor to GPlb on these platelets.

of this syndrome. In the other family an A to G mutation leads to a valine for methionine substitution in position 239 (Russell and Roth, 1993). These close mutations lie in a 40 amino acid, disulfide-bridged loop thought to form part of the vWf binding site (Hess et al., 1991). It seems likely that changes in the exposure or conformation of this loop caused by high shear conditions are important for determining at least part of the interaction with vWf in physiological primary hemostasis. Other mutations in this critical region may be predicted to give rise to similar defects or, to the opposite effect, a reduced binding of vWf to GPIb. Figure 7 shows a schematic drawing of the double loop region of GPIba with the platelet-type von Willebrand disease mutation sites indicated. The equivalent disorder caused by mutations in vWf is Type IIB von Willebrand’s disease (De Marco et al., 1985). Such mutations have been localized to the region of vWf identified as the GPIb binding site (Cooney et al., 1991; Randi et al., 1991). Thus, changes in the conformation of either the vWf binding site on GPIb or the GPIb binding site on vWf can lead to binding of one to the other pointing to a role for such changes in the physiological functioning of the GPIbIvWf axis. B. Biochemical Evidence for Function

Considerable biochemical evidence for the localization of the vWf binding site on GPIb has been accumulated. This was first of all based on both polyclonal and monoclonal antibodiesthat block vWf-related platelet function and that were found to bind to GPIb (Ali-Briggs et al., 1981; Coller et al., 1983; Ruan et al., 1981). The

44

KENNETH J.CLEMETSON

epitopes could be further localized to the N-terminal45 kDa domain of GPIb but since they are conformation dependent could not yet be more precisely determined. Treatment of platelets with proteases, such as calpain or trypsin, that preferentially remove glycocalicin, the major part of the extracellular domain of GPIba, or with proteases, such as elastase (Brower et al., 1985; Wick and Clemetson, 1985), that selectively remove the 45 kDa N-terminal domain both lead to platelets that no longer bind to vWf also supporting the localization of the vWf-binding domain in this region. Further, both recombinant fragments (Cruz et al., 1992) and peptides derived from the GPIb sequence have been used as competitive inhibitors of the platelet/vWf interaction in attempts to localize the binding site more precisely (Vicente et al., 1990; Katagiri et al., 1990). Since vWf does not normally interact directly with GPIb it was necessary to induce binding using either ristocetin, botrocetin, or asialo vWf. It was first of all established that the disulfide bonds of the 45 kDa domain were necessary for optimal interactions except that induced by ristocetin. Then, in one case 27 overlapping peptides covering the 45 kDa domain were used in inhibitory assays (Vicente et al., 1990). The sequence Ser251-Tyr279 was identified as inhibiting ristocetidvWf-induced platelet agglutination but also inhibited botrocetin-induced platelet agglutination at higher concentrations. In both cases very high amounts of peptide (about 0.5 mM) were necessary. It should be noted that this peptide contains most of the smaller double loop (in linear form) plus th’e anionic region in the C-terminal direction and also that it contains a PG sequence (see below). The other study reported the peptide Asp235-Lys262 (which still contains the PG sequence) to be capable of inhibiting ristocetidvWf-induced platelet aggregation at 11 pm whereas the peptide Asp249-Asp274 (which also containsthe PG) required 300 pm for a comparable inhibition(Katagiri et al., 1990). Lastly, site directed mutagenesis has been used to try to establish residues in GPIba critical for vWf binding (Ruggeri). Thus, conversion of aspartic acid and glutamic acid rezidues to asparagine and glutamine, respectively, in the region between 25 1 and 279 elisinated binding to vWf in the presence of either ristocetin or botrocetin whereas substitutions between 280 and 302 only affected botrocetin. However, it remains unclear how such mutations affect folding and disulfide bond formation in mutant proteins so that alternative explanations remain open.

C. The Binding Site for GPlb on vWf The structure of vWf is also broadly known although many of the details remain to be determined. It has been known for some time that the A1 domain contains sites for GPIb, collagen, and heparin interactions (Girma et al., 1987; Mohri et al., 1989) and more recent studies of sequence mutants, Type IIb von Willebrand’s disease, the effects of glycosylation and differences with animal vWf have narrowed down the GPIb binding site to a disulfide loop formed by a cysteine bridge and neighboring sequences (see Azuma et al., 1993; Cooney et al., 1991; Fujimura

platelet Adhesion Receptors

45

et al., 1986; Girma et al., 1990; Handa et al., 1986; Randi et al., 1991; Sixma et al., 1991; Sugimoto et al., 1991). The fact that a collagen-binding site is in close proximity supports the idea that conformational changes in the GPIb-binding site are probably induced by collagen-binding.

D. Non-physiological Activators of the GPlb/vWf Axis Since vWf in plasma does not normally interact with GPIb and it has so far been difficult to assemble a simple, in vitro, system that accurately reflects the way in which vWf is activated in the subendothelium, various non-physiological methods have been used to induce vWf/GPIb interactions. The simplest of these has been to use either animal vWf (generally bovine or porcine) (Cooper et al., 1979; Kirby, 1982) or human vWf which has been treated with neuraminidase to remove sialic acid (De Marco and Shapiro, 1981; Gralnick et al., 1985). Presumably all of these agglutinate human platelets because, due to differences in sequence in the case of the animal vWf or to changes in conformation caused by removal of the sialic acid, they are already in a conformation favorable for binding of GPIb. Since we do not yet have X-ray crystallographic data on the appropriate fragments of vWf and GPIb the interactions involved remain unclear. Other reagents have been discovered that are capable of inducing interactions between normal human vWf and platelet GPIb. These include ristocetin and botrocetin (Howard et al., 1984; Girma et al., 1990). Ristocetin has been known for a number of years (Howard and Firkin, 1971) and has been useful for diagnosis of bleeding disorders related to the GPIb/vWf axis (Zucker et al., 1977). It is a glycopeptide antibiotic which is thought to act by binding D-ala containing peptides and thus preventing cross-linking within the peptidoglycans forming the growing cell wall of bacteria. How it induces the vWflGPIb interaction is still controversial but it is thought to bind to both vWf and GPIb (Sixma et al., 1991). Aplausible explanationextending earlier ideas (Jenkins et al., 1979) suggested that dimeric ristocetin can link the two molecules and indicated that XPGX sequences in both were important for binding (Scott et al., 1991). Others have pointed to XPPX sequences in vWf as being important (Azuma et al., 1993). It has been suggested that vWf-GPIb binding involves electrostatic interactions and that ristocetin may tip this delicate balance but simple cross-linking via dimeric ristocetin would also seem to be adequate. Botrocetin is a peptide mixture from the venom of the snake Bothropsjururucu (Read et al., 1978; Andrews et al., 1989) that interacts with von Willebrand factor and induces it to bind to GPIb on platelets. Recently, the most active species was shown to be a two-chain molecule consisting of a- (1 5 kDa) and p- (14.5 kDa) subunits linked by disulfide bonds (Usami et al., 1993).The other less active species consists of a single peptide chain of 27 kDa apparently unrelated in sequence to the other chains. Structural analysis indicates a strong similarity to, among others, C-type (Ca2+dependent) lectins, however, the effect on vWf was not inhibited by EDTA or a variety of sugars (Usami et al., 1993). Studies with peptides and

46

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site-directed mutagensis indicate that botrocetin binding to vWf occurs via several segments of the A1 loop. It should be noted that human vWf that has been treated with neuraminidase to remove sialic acid (asialo vWQ also binds spontaneously to platelet GPIb. Although this has been presented as a charge effect (sialic acid carries most of the negative charge on GPIb and on the platelet surface) the fact that further treatment to remove galactose residues makes the vWf again unresponsive would rather argue for an important role of oligosaccharidesin the conformationalchanges in vWf. Since the mode of action of ristocetin and botrocetin are clearly distinct it should not be surprising that different regions of vWf (Sugimoto et al., 1991) and GPIb are involved (Girma et al., 1990) and it is certainly valid to wonder to what degree they really simulate the physiological mechanism. They have, nevertheless, proved to be very useful tools for investigating many aspects of this process. Recently,some other snake peptides have been discoveredthat affect the vWf7GPIb axis. Alboaggregin-B from Trimeresus albolabris has been shown to induce platelet agglutination to vWf by binding to GPIb (Peng, 1993) whereas echicetin from Echis curinatus, aglucetin from Agkistrodon acum (Chen et al., 1995), tokaracetin from Trimeresum tokarensis (Kawasaki et al., 1999, flavocetin-A and -B from Tnmeresurusflavoviridis (Taniuchi et al., 1995), and jararaca GPIb-BP from Bothmpsjararucu (Kawasaki et al., 1996) blocks platelet agglutination by attachment to the same site (Peng et al., 1993). It is interesting that alboaggregin-B and botrocetin have very similar sequences (Yoshida et al., 1993) and yet such apparently dissimilar mechanism providing an interesting example of evolutionary adaptation. A consideration of all these various examples allows the construction of a model for the physiologicalprocess. Although it is not yet possibleto test all the parameters involved there is some evidence for most of them. Thus, the presence of vWf is necessary on the subendothelium and it must be associated with specific components in order to undergo a conformational change so that it can bind to GPIb on the platelets. The prime candidate is collagen although microfibrils containing proteoglycans have also been proposed (Fauvel et al., 1983). The domain of vWf containing the GPIb binding site also has a collagen binding site in close proximity (Girma et al., 1986; Sixma et al., 1991) which could well be involved in the conformational change. However, the results obtained from the studies of both genetic disorders and non-physiologicalreagents imply that it is possible to enhance the vWf/GPIb interaction from both sides implying that changes in GPIb may also be involved in the physiological process. If the problem of primary hemostasis is considered it is clear that platelets need to adhere to subendothelium under a wide range of conditions and these two parameters, changes in both vWf and in GPIb may provide the necessary flexibilityto handle this. At high shear rates in particular it is important that the platelets touching the subendothelium are stopped rapidly and maintained in intimate contact with the surface so that hrther processes can be activated. Probably an important factor is that many binding sites are involved simultaneously and this is why the multimeric form of vWf and its alignment on collagen fibers may be important. Here also, the large number of GPIb molecules

Platelet Adhesion Receptors

47

on the platelet surface and their distribution may be important. Having stopped the platelet and brought it into contact with the subendothelium it is necessary to activate it and have it spread so as to cover a wide surface and so involve the maximum of interactions with adhesive proteins in the subendothelium. This activationprocess may be started by the interaction of GPIb and vWf, together with the forces exerted on the platelet by shear. Evidence for signaling via GPIb will be discussed later. It may also be caused by the interaction of other receptors with adhesive proteins, in particular with collagen (see below). Finally, following activation there are a wide number of changes in the platelet involving changes in other receptors and their relationship with the cytoskeleton and also the release of a-granules and incorporation of their membrane glycoproteins into the plasma membrane that all affect the sum of the adhesion process.

E. The Role of the GPlb-V-IX Complex in Thrombin Activation of Platelets The GPIb-V-IX complex contains two thrombin interactive sites, one on GPIba and the other on GPV. The site on GPIba was shown to bind thrombin at an early stage in the characterization of glycocalicin (Okumura et al., 1978). In BSS, where GPIb is absent, the platelets show a reduced response to thrombin which can be reproduced by treating platelets with enzymes that remove selectively the outer domains of GPIba or by antibodies (polyclonal or monoclonal) that recognize the thrombin binding site on GPIba (Jenkins et al., 1983; Mazurov et al., 1991; De Marco et al., 1991). More detailed analysis showed that removal or blockage of this site only affected the platelet response to low doses of thrombin (Wicki and Clemetson, 1985; McGowan and Detwiler, 1986)and had little effect at high doses. This was only true for a-thrombin because y-thrombin showed the same slow kinetics with plateletswhether or not GPIb was present (Jandrot-Permset al., 1990). It is also known that y-thrombin does not bind to GPIb (Jandrot-Permset al., 1988). The thrombin receptor, discussed by Coughlin in Chapter 5 , is now known to belong to the seven transmembrane, G-protein coupled receptor family and to be the first known representative of a mechanism where proteolytic cleavage of the N-terminus of the receptor reveals a new N-terminus which acts as a “tethered ligand” capable of interactingwith other extracellular loops to activate the receptor (Vu et al., 1991). In addition it was also shown that the thrombin receptor N-terminus contains a highly charged domain similar to the C-terminal region of hirudin capable of binding to the anion-bindingregion of thrombin and thus facilitating the interaction between thrombin and the receptor (Liu et al., 1991). GPIb contains a region just on the C-terminal side of the double-loop which shows some similarity to this domain (Jandrot-Perrus et al., 1992a, 1992b). However, an N-terminal domain containing the double-loop region but with most of the charged domain removed, bound both thrombin and vWf more avidly than the fragment containing the complete highly charged domain (Kresbach et al., 1991) casting doubt on a role for this domain in thrombin (and vWQ binding. Two theories have been proposed for

KENNETH J. CLEMETSON

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the role of GPIb in thrombin-activation of platelets, which may play a role in the post-initial-adhesion activation and spreading of platelets on subendothelium.One of these suggests that the thrombin receptor is in close proximity to GPIb on the platelet surface and that the binding site on GPIb first of all alters the conformation of thrombin to make the binding site more accessible and also aids in docking and directing the active site of the thrombin to the cleavage site of the receptor. Indeed, it is known that the active site of thrombin is not blocked by binding to GPIb. An open question here is whether the hirudin-like sites on GPIb and the thrombin receptor are competitive or not. In order to obtain a synergistic effect it would be necessary to have simultaneous binding of thrombin to both GPIb and the receptor. Another problem with this theory is the large number of GPIb molecules (25,000) in comparison with the thrombin receptor (1,200). Of course, a close proximity of the two sites might account for the “high avidity binding site”(Grec0 and Jamieson, 1991) by increasing the avidity for thrombin through simultaneous binding. Experiments with coexpression may clarify the relationship between the GPIb complex, the thrombin receptor and thrombin. An alternative explanation for the role of GPIb in thrombin-inducedplatelet activation might be related to signal transduction via GPIb to the platelet interior. It should immediately be noted that there is no direct evidence for such a signal but the phenomenon of priming is known from other cells. It should also be noted that the function of GPIbP, GPIX and GPV still remains obscure. As mentioned above GPIbP contains a site that can be phosphorylated by CAMPdependent kinase and which may play a role in regulation of the cytoskeleton (Fox and Berndt, 1989). It remains conceivable that thrombin, in binding to GPIb, causes a conformational change that affects the cytoplasmic domains and facilitates signal transduction from the thrombin receptor, particularly during the early stages. To elucidate how such a system might work it will be necessary to have a better understanding of how the thrombin receptor itself causes signal transduction. Although much progress has been made there are still gaps in our knowledge especially in understanding how the kinetics of the system are controlled. Here again, co-expression studies may be expected to help. F. Expression of GPlb-V-IX

Since very little is known about how the various chains of the complex interact with each other and with other molecules (such as in the cytoskeleton), it is of considerable interest to be able to express these in various combinations on non-megakaryocytic cells. Two studies have appeared, one reporting that expression of all three (Iba,IbP, and IX)chains is necessary for an efficient overall expression of any (Lopez et al., 1992) while the other found that Iba by itself although extensively degraded intracellularly could be expressed intact in small amounts (Meyer et al., 1993).Co-expression ofGPV was also demonstrated to give an association with GPIb and to enhance expression of the other components (Li et al., 1995; Caverly et al., 1995).

platelet Adhesion Receptors

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G. Platelet Activation and Signaling Transduction Via the GPlb/vWf Axis We have seen that after the initial adhesion of the platelet to the subendothelium it is activated to bring about the necessary changes to stabilize the interaction and to initiate repair processes. A controversial point for some time was the question whether the binding of GPIb to vWf could itself induce transduction of signals. For many years the phenomenon of platelet-platelet binding induced by ristocetin in the presence of vWf was referred to as agglutination precisely to make the point that this was indeed a passive process with no activation of the platelets involved. However,more recent results exposing platelets to shear-forcescomparable to those encountered under more rigorous physiological conditions indicate that the GPIb/vWf axis is indeed capable of causing a weak signal, seen as a rise in calcium in the platelets (Kroll et al., 1991, 1993; Chow et al., 1992;Ikeda et al., 1993).How such signals are induced and how they cause hrther platelet activation remain unclear but it has been suggested that it is the force on the GPIb-V-IX complex, transmitted to the cytoskeleton, which is important for opening Ca2+channels. Where high shear force is not present there is little evidence for a direct signal through GPIb after adherence to subendothelium. However, the primary adhesion process in itself brings other receptors into action such as the collagen receptor@).

V. OTHER ADHESION RECEPTORS A. Collagen Receptors

Although collagen receptors are dealt with in detail elsewhere (Chapter 4, Santoro, Saelman, and Zutter) their role in adhesion and cross-talk with other receptors needs to be placed briefly in context. Collagen is not the only substrate involved in this secondary phase of adhesion, since fibronectin, laminin, and thrombospondin are all present in the subendothelium and the platelet has receptors for all of these, but it is undoubtedly the most important because of its prevalence, variety, and its strong platelet activating properties. Over the year a very large number ofplatelet molecules have been proposed as collagen receptors with widely varying amounts of evidence. These have now been reduced to only a few. There is good evidence for GPIa-IIa (a2&) as a major collagen receptor (Santoro et al., 1988) based on studies with patients whose platelets have defects in this integrin complex (Nieuwenhuis et al., 1985; Kehrel et al., 1988) and also on the use of specific antibodies (Staatz et al., 1989; Coller et al., 1989). The structure of this complex is shown in Figure 8. In platelet adhesion, when GPIa-IIa is not functional, the platelets remain poorly associated with the subendothelium, with only a few points of attachment and are not activated or spread, probably reflecting mainly the adhesion through the GPIb/vWf axis (Nieuwenhuis et al., 1986). Other platelet glycoproteinshave also been implicated as collagen receptors though their precise roles remain uncertain. These include CD36 (GPIIIb or GPIV) which was shown

KENNETH J. CLEMETSON

50

-DGEA-

GPla

a2 esium-binding sites

Phosphorylationsite

Cytoplasmic domains

Figure 8. Schematic drawing of GPla-lla (a&). Unlike GPllb-llla this integrin is constitutively active and the role of the phosphorylation sites on the cytoplasmic domain of p1 remains obscure. The divalent cation binding sites prefer magnesium or manganese for optimal activity of the complex and calcium has an inhibitory effect on collagen binding.

to bind collagen in in vitro tests. CD36 has been cloned (Oquendo et al., 1989) and was the first representative described (Figure 9) of a new family of membrane proteins. However,apparently normal individuals lacking CD36 (Naka-phenotype) do not have hemostatic problems (Yamamoto et al., 1992) and their platelets show minor differences in collagen reactivity (Tandon et al., 199lb), reduced adhesion to collagen in flowing blood (Diaz-Ricart et al., 1993), an increased reactivity with collagens I and I11 and a lack of response to collagen V (Kehrel et al., 1993; McKeown et al., 1993). Collagen V is only involved in platelet adhesion in static or low shear situations but occurs in increased amounts in atherosclerotic plaque so the significance of these findings is still not obvious. Platelet aggregation to collagen was inhibited by an antibody against an 85- to 90-Kd platelet glycoprotein in a patient with prolonged bleeding time (Deckmyn et al., 1992).It remains unclear if the molecule recognized is CD36 or not. Platelet GPVI has also been described as a further receptor for collagen based on studies with one patient apparently lacking this glycoprotein (Ryo et al., 1992)and on another with antibodies directed against a similar glycoprotein (Moroi et al., 1989). Recently, GPVI was shown to be implicated in the activation of c-src and ~ 7 2 tyrosine ’ ~ ~ kinases (Ichinohe et al., 1995). Since many kinds of collagen exist and the circumstances where collagen

Platelet Adhesion Receptors

51

Proline-rich

Membrane Cytoplasm pp60src-relatedkinases Fyn, Yes, Lyn, Hck V Potential N-glycosylation sites

I

CXCBXBXXK seauence B = basic amino'acid

Figure 9. Schematic drawing of CD36 (GPlllb or IV) showing putative domains and interaction sites with cytoplasmic tyrosine kinases of the src family.

receptors might play a role are varied it cannot be excluded that these other receptors may have a modulatory function or that complexes of these glycoproteins are involved. B. Fibronectin and Larninin Receptors

The role of fibronectin receptors (GPIc-IIa = aspI) and laminin receptors p67) in adhesion remains uncertain (Piotrowicz et al., 1988; (GP1c'-IIa = a6pI; Parmentier et al., 1991) despite some indication that they may play a supportative role. Thus, one patient who was reported to have a GPIIa deficiency (unpublished) leading to a lack of all of this class of integrins on the platelets had more severe bleeding problems than those patients with simply GPIa-IIa deficiencieswhere the other integrins are normal. Other laminin receptors have also been described, in particular a 67 kDa glycoprotein (Tandon et al., 1991a; Hindriks et al., 1992). C. Thrornbospondin and Its Receptors in Adhesion

Recent studies have clearly indicated that the Ca2+form of thrombospondin can support platelet adhesion (Agbanyo et al., 1993; Tuszynski 8z Kowalska, 1991) although the platelet receptors involved in this process are not yet well defined. As with collagen there are a number of possible candidates, the best known of which are CD36 and GPIIb-IIIa and, possibly GPIa-IIa. However, Nak"- platelets, where CD36 is missing, bind thrombospondin at normal levels (Kehrel et al., 1991;

KENNETH J. CLEMETSON

52

Tandon et al., 199lb) implying that platelets have other classes ofreceptors possibly related to heparin. D. The Vitronectin Receptor

The vitronectin receptor on platelets is closely related to the fibrinogen, GPIIbIIIa, consisting of an a,-chain integrin together with the IIIa (p3) chain. There are between 1,500-5,000 copies per platelet which may be enough to account for some GPIIIa expression found in Glanzmann’sthrombasthenia platelets where the GPIIb gene is affected. The role of the vitronectin receptor in adhesion and aggregation is still controversial with suggestions that it may be able to substitute partly for GPIIbAIIa while others have shown that vitronectin can inhibit fibrinogen binding to platelets.

E. GPllb-llla in Adhesion While the major role of the integrin GPIIb-IIIa (arIbP3) is clearly in platelet-platelet aggregation there is considerable evidence that it also plays an important role in adhesion. In addition to binding fibrinogen it has also been shown to bind fibronectin (Parise and Phillips, 1986; Plow et al., 1985), von Willebrand factor (Plow et al., 1985) and possibly thrombospondin (Karczewski et al., 1989) and vitronectin (Mohri and Ohkubo, 1991). Thus, Glanzmann’s thrombasthenia platelets which are deficient in GPIIb-IIIa show a reduced adhesion to subendothelium, though not as dramatic as in BSS when GPIb is missing. Studies with various antibodies point to two effects here. One is that platelets, which have adhered and have been wrenched from the subendothelium by shear, are activated and can bind through GPIIb-IIIa to vWf-coated exposed subendothelium downstream of the initial adhesion site (Ruggeri et al., 1983). The second is that GPIIb-IIIa is apparently necessary for the spreading of the adhering platelets on subendothelium and Glanzmann’s thrombasthenia platelets show a much reduced contact area compared with normal (Lawrence and Gralnick, 1987). This role can be explained by activated GPIIb-IIIa binding to vWf and other adhesive proteins on the vessel wall (Savage et al., 1992) and, thus, increasing the association between the platelet cytoskeleton and the subendothelium but also by the important function of GPIIbIIIa in signal transduction via tyrosine kinases and phosphatases and in activation of pathways involved in cytoskeletal rearrangement (Ferrell and Martin, 1989; Golden et al., 1990; Kieffer et al., 1992; Haimovich et al., 1993). In the absence of GPIIb-IIIa or if it is blocked, these cytoskeletal changes are prevented or severely reduced thus preventing spreading from occurring efficiently. Recent studies have also shown that GPIIb-IIIa in the unactivated state is also the receptor for surfacebound fibrinogen (Zamarron et al., 1991). Whether this may also mediate interactions between platelets and subendothelium appears unlikely but cannot be completely excluded.

Platelet Adhesion Receptors

53

VI. INHIBITION OF PLATELET ADHESION AS A PROPHYLACTIC MEASURE OR FOR TREATMENT OF ACUTE THROMBOTIC EVENTS Platelet aggregation has been a prime target for the development of inhibitory drugs for treatment of acute thrombotic events such as restenosis and eventually for prophylaxis against thrombosis and several products are in advanced stages of testing. Since the long term role of aggregation versus adhesion in atherosclerotic processes or even in acute events is still virtually unknown, it is worth developing alternative strategies to inhibition of aggregation and the complex process of adhesion presents an attractive target. Clearly, the GPIb/vWf axis is the best understood (if still only partially) of these mechanisms. Methods based upon peptides from the GPIb binding region of vWf or from the vWf binding region of GPIb could be used as a first approach to this in the same way that RGD-containing peptides were used or snake venom peptides were used as the first approach to blocking aggregation. The availability of snake venom peptides capable ofblocking the GPIb-vWf interaction (Peng et al., 1993) may also provide a usefbl starting point. Just as anti-GPIIb-IIIa/fibrinogen drugs simulate the situation in Glanzmann’s thrombasthenia, anti-GPIb/vWf drugs should simulate, at least partially, BSS. Some examples have already been reported where the GPIb-binding domain of vWf in recombinant form was demonstrated to inhibit platelet adhesion to extracellular matrix (Dardik et al., 1993; Prior et al., 1993) and a recombinant fragment of GPIba has been shown to inhibit vWf binding to GPIb and also to collagen. The IC,, was, however, 4 pM, which is nevertheless high compared to RGDS (1C5, 0.1 pM)by no means the most efficient of this class of inhibitors. It is, therefore, worth considering how hemostasis operates in such patients and why bleeding only occurs normally as a consequence of major trauma, since one of the major side effects of such treatments might indeed be major bleeding episodes. In fact it is quite surprising in view of the apparently critical roles of both GPIIb-IIIa and GPIb-V-IX in hemostasis that bleeding episopes are so restricted. If the lack of IIb-IIIa is considered then adhesion is normal and a damaged surface is quickly protected. The main problem comes from a lack of clot retraction drawing together the sides of a wound and the inability to block sectioned vessels quickly. This is also a consequence of the lack of anchors for the fibrin net that normally strengthens a thrombus. However, since platelets can adhere, are activated and do secrete granule contents, most of the reparative processes can occur normally. In the case of BSS it might be expected that the situation would be much worse and indeed, on the whole, BSS patients do have more problems. Many, however, live relatively normal lives if they avoid hazards. It must therefore be assumed that even in the absence of GPIb-V-IX (which is often still present albeit in very small amounts) enough platelets adhere to the subendothelium to mediate hemostasis. Here, binding of activated GPIIb-IIIa to vWf might substitute partly for the GPIb/vWf axis and once a few platelets adhere and are activated they can bind and activate resting

54

KENNETH 1. CLEMETSON

platelets arriving with the blood stream. Alternatively, the large size and easy deformation of BSS platelets may play a role in allowing enough adhesion to occur via other receptors to cover the damaged site. Spreading of activated BSS platelets should not present any noticeable problems. Although these postulated compensatory mechanisms are either not present or function differently on normal platelets in the presence of drugs it will be necessary to explore dose-response very carehlly to see if side effects are a problem. It should also not be forgotten that individuals differ widely in activity of many factors and a mild coagulation defect that is not apparent under normal conditions may cause problems when platelet adhesion is inhibited.

VII. ADHESION OF PLATELETS TO OTHER CELLS As well as adhering to subendothelium and to other platelets, adherence of platelets to other cells may well be of great physiological importance. These cells include neutrophils and monocytes (McEver, 1991; Rinder et al., 1991;) but under special circumstances platelets can also adhere to endothelial cells (Etingin et al., 1993). These various interactions are partly dependent on some of the receptors already described above such as GPIb and GPIIb-IIIa but mainly involve the expression of new receptors on activated platelets from the membranes of the platelet storage granules. A. P-Selectin (CD62, GMP-140, PADGEM)

This is a granule membrane glycoprotein with a molecular mass of 140 kDa (hence GMP- 140). PADGEM is derived from Platelet Activation Dependent Granule External Membrane protein (Hsu-Lin et al., 1984). Out of a total of 789 amino acids, from the N-terminus the structure (Figure 10) contains a 120 amino acid lectin-like domain, a 40 amino acid epidermal growth factor-like domain, nine 62 amino acid repeats similar to complement-binding protein, a transmembrane domain, and a 35 amino acid cytoplasmic domain (Johnston et al., 1989). An alternatively spliced message for P-selectin contains no transmembrane domain and would be predicted to code for a soluble form (Ushiyama et al., 1993). Similar structures have been found on other cells (endothelial leukocytes and leukocytes) and the name selectins was proposed for the family (from the lectin-like domain) with the prefix letter designating the cell-type (e.g., P- for platelet). In resting platelets P-selectin is found in the membrane of the a-granules and in endothelial cells in equivalent structures, the Weibel-Palade bodies (McEver et al., 1989; Bonfanti et al., 1989). After platelet stimulation with agonists such as thrombin the release reaction from the granules occurs and the P-selectin, together with other granule membrane constituents, is transferred via membrane fusion to the plasma membrane. While resting platelets express 1000 or less P-selectin molecules when they are activated this rises to about 10,000. This exposed P-selectin can then bind

55

Platelet Adhesion Receptors

CD62 P-SELECT1N

oplasmic domains 3;’ P osphorylationsites figure 10. Schematic drawing of P-selectin (CD62) and of PECAM-1 (CD31) showing domain structure and homologies to related proteins.

to carbohydrate structures ofthe sialyl Lewis’ and sialyl Lewisaclass on glycolipids or glycoproteins of neutrophils and myeloid cells (Erbe et al., 1993; Larsen et al., 1990) and may be involved in elimination of activated platelets from the circulation on one hand or in binding neutrophils to a platelet thrombus on the other. Since there is considerable evidence for certain complementary activities between activated platelets and neutrophils in, for example, inflammatory loci, this may also be a way of maintaining the necessary interactions. The equivalent molecule on endothelial cells seems to play an important role in the phenomenon of leukocyte “rolling,” where leukocytes are held in loose contact with the endothelial surface but are, nevertheless, moved along by the blood flow. This can also occur via P-selectin on a platelet layer (Buttrum et al., 1993). P-selectin is rapidly phosphorylated on serine, threonine, and tyrosine when platelets are activated but phosphothreonine and phosphotyrosine are rapidly dephosphorylated leaving only phosphoserine after five minutes (Crovello et al., 1993). The function of this phosphorylation is still unknown. P-selectin is acylated with palmitic acid and stearic acid at cysteine 766 through a thioester linkage (Fujimoto et al., 1993).

KENNETH J. CLEMETSON

56

6. PECAM-1 (Platelet and Endothelial Cell Adhesion Molecule, 0 3 1 ) This molecule (130 kDa) is a platelet adhesion receptor but is also found on endothelial cells, neutrophils and monocytes (Newman et al., 1990). PECAM-1 is a highly glycosylated molecule with 40% carbohydrate (Figure 10). It shows similarities to both the Fc portion of IgG and to carcinoembryonic antigen. Six Ig-like domains are present. The C-terminal region contains both a transmembrane domain and a long serine- and threonine-rich cytoplasmicdomain. Phosphorylation of this domain seems to be important for regulating the activity of the molecule. PECAM- 1 is rapidly phosphorylated on serine residues after platelet activation and becomes associated with the platelet cytoskeleton (Newman et al., 1992). The role of PECAM-1 in platelet function is still obscure. It seems certain that it is not involved directly in platelet aggregation, implying that it has an adhesive function either to subendothelial components or, more likely, to other cells. Recent results have demonstrated that possible ligands for PECAM in heterotypic adhesion may be cell surface glycosaminoglycans (DeLisser et al., 1993)and a consensusbinding sequence, LKREKN, present in the second immunoglobulin-like homology domain was shown to be involved.

ACKNOWLEDGMENTS Support for the work described here camed out at the Theodor Kocher Institute, from the Swiss National Science Foundation Grant 31-32416.91, by a grant from Hoffmann-La Roche Ltd., and by the supply of b u m coats from the Central Laboratory of the Swiss Red Cross Blood Transfusion Service, is gratefilly acknowledged.

REFERENCES Agbanyo, F. R., Sixma, J. J., De Groot, P. G., Languino, L. R., & Plow, E. F. (1993). Thrombospondinplatelet interactions. Role of divalent cations, wall shear rate, and platelet membrane glycoproteins. J. Clin. Invest. 92,28%296. Ali-Brigs, E. F., Jenkins, C. S. P., & Clemetson, K. J. (1981). Antibodies against platelet membrane glycoproteins: Crossed immunoelectrophoresis studies with antibodies that inhibit ristocetin-induced platelet aggregation. Br. J. Haematol. 48, 305-3 18. Andrews, R. K., & Fox, J. E. B. (1991). Interaction of purified actin-binding protein with the platelet membrane glycoprotein Ib-IX complex. J. Biol. Chem. 266,7144-7147. Andrews, R. K., Booth, W. J., Gorman, J. J., Castaldi, P. A., & Berndt, M. C. (1989). Purification of botrocetin from Bothrops jararaca venom. Analysis of the botrocetin-mediated interaction between von Willebrand factor and the human platelet membrane glycoprotein Ib-IX complex. Biochemistry 28,8317-8326. Azuma, H., Sugimoto, M., Ruggeri, 2. M., & Ware, J. (1993). A role for von Willebrand factor proline residues 702-704 in ristocetin-mediated binding to platelet glycoprotein Ib. Thromb. Haemost. 69, 192-1 96. Bennett, J. S. (1990). The molecular biology of platelet membrane proteins. Semin. Hematol. 27, 186-204. Berndt, M. C., Gregory, C., Chong, B. H., Zola, H., & Castaldi, P. A. (1983). Additional glycoprotein defects in Bernard-Soulier’s syndrome: Confirmation of genetic basis by parental analysis. Blood 62,8004307.

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