IUBMB
Life, 56(1): 13–18, January 2004
Review Article Platelet Interactions in Thrombosis Robert K. Andrews, Elizabeth E. Gardiner, Yang Shen and Michael C. Berndt Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168, Australia
Summary Patho/physiological platelet aggregate (thrombus) formation is initiated by engagement of platelet surface receptors, glycoprotein (GP)Ib-IX-V and GPVI that bind von Willebrand factor or collagen. Although beneficial in response to vascular injury by preventing blood loss (haemostasis), platelet aggregation in a sclerotic coronary artery or other diseased blood vessel (thrombosis) can cause thrombotic diseases like heart attack and stroke. At the molecular level, ligand interactions with GPIb-IX-V or GPVI trigger signalling responses, including elevation of cytosolic Ca2 + , dissociation of calmodulin from their cytoplasmic domains, cytoskeletal actin-filament rearrangements, activation of src-family kinases or PI 3-kinase, and ‘inside-out’ activation of the integrin, aIIbb3 (GPIIb-IIIa), that binds von Willebrand factor or fibrinogen and mediates platelet aggregation. Furthermore, emerging evidence supports a topographical coassociation of these receptors of the leucine-rich repeat family (GPIb-IX-V) and immunoglobulin superfamily (GPVI) in an adhesive cluster or ‘adhesosome’. This arrangement may underlie common mechanisms of initiating thrombus formation in haemostasis or thrombotic disease. IUBMB Life, 56: 13–18, 2004 Keywords Thrombosis; platelets; adhesion receptors; GPIb-IX-V; GPVI
INTRODUCTION Platelet aggregate or ‘thrombus’ formation in response to vascular injury or disease is initiated by two unique membrane-surface receptors: glycoprotein (GP)Ib-IX-V and GPVI. GPIb-IX-V binds the multimeric adhesive glycoprotein, von Willebrand factor, secreted by activated platelets or endothelial cells, and present in blood plasma or subendothelial matrix (1, 2). GPVI binds collagen (3). GPIb-IX-V and GPVI initiate platelet aggregation at high or low hydrodynamic shear stress, respectively. In spite of their common Received 1 October 2003; accepted 14 November 2003 Address correspondence to Robert K. Andrews, Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3168, Australia. E-mail:
[email protected] ISSN 1521-6543 print/ISSN 1521-6551 online # 2004 IUBMB DOI: 10.1080/15216540310001649831
function, these receptors are structurally unrelated. GPIb-IXV is a complex of four type-I transmembrane subunits of the leucine-rich repeat family: GPIba, GPIbb, GPIX and GPV in the ratio 2 : 2 : 2 : 1 (4). Each subunit contains one or more leucine-rich repeat domains in the extracellular region. GPVI on the other hand is a member of the immunoglobulin superfamily, and consists of a type-I transmembrane glycoprotein containing two immunoglobulin domains in the extracellular region (3). Recent studies of the function and signalling responses of GPIb-IX-V and GPVI, however, have revealed a number of common properties, and raise the possibility that these receptors may be co-associated on the platelet surface. This review will discuss recent findings on structure-function relationships and signalling mechanisms of GPIb-IX-V and GPVI, and speculate on whether this emerging evidence implies a functional co-association of these distinctive receptors.
GPIb-IX-V The physiological importance of the platelet membrane GPIb-IX-V complex was initially inferred from the rare inherited human bleeding disorder, Bernard-Soulier syndrome (5). A dysfunctional receptor complex results from one of many reported lesions of GPIba, GPIbb or GPIX, but not GPV, genes (4, 5). Bernard-Soulier syndrome often results in severe bleeding, and is associated with defective adhesion of a patient’s platelets to von Willebrand factor at high hydrodynamic shear flow. Thrombin-dependent aggregation is also delayed in Bernard-Soulier syndrome platelets, whereas response to other agonists such as collagen and ADP is largely unaffected. In virtually all cases of Bernard-Soulier syndrome, abnormal GPIb-IX-V results in large spherical platelets. This implies a role for GPIb-IX-V in maintaining normal platelet size and biconcave discoid shape. In a mouse model of Bernard-Soulier syndrome, where platelets are deficient in wild-type GPIb-IXV, introduction of just the cytoplasmic portion of GPIba as part of a chimera with the IL-2 receptor extracellular domain
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substantively rescued the large-platelet phenotype (6); without the extracellular von Willebrand factor-binding domain of GPIba, however, the mice still bled abnormally. These and other studies have revealed a two-way regulatory relationship between GPIb-IX-V and the platelet cytoskeleton (1, 7 – 11). One of the ways GPIba can potentially regulate cytoskeletal actin is by sequestering the signalling protein, 14-3-3x (11), which binds to a site at the C-terminus of GPIba that includes the constitutively-phosphorylated residue, serine-609 (12). 143-3x regulates Rho/GTPases that control actin polymerization. Cell expression of a full-length GPIba cytoplasmic domain (fused to the extracellular domain of the IL-2 receptor), but not a truncated GPIba tail lacking the 14-33x-binding site, inhibited integrin-mediated cytoskeletal changes (11). An upstream sequence within the GPIba cytoplasmic domain binds actin-binding protein (filamin), which links GPIb-IX-V to submembranous, short actin filaments (membrane skeleton) in resting platelets. Truncation, deletion or point mutations of the GPIba cytoplasmic domain that disrupt filamin binding clearly modulate von Willebrand factor binding to the extracellular portion of GPIba. Surprisingly, however, the interaction with von Willebrand factor is either increased or decreased, depending on how filamin association is disrupted, expression levels, and/or the analytical conditions used (7 – 10). This is an interesting phenomenon, and may imply that cytoskeletal attachment to the receptor could differentially regulate platelet adhesion and activation (and vice versa), depending on the patho/physiological context. In a similar manner, mutation of serine-166 of GPIbb, a protein kinase A (PKA)-dependent phosphorylation site within another 14-3-3x-binding motif, also differentially regulates von Willebrand factor binding under different experimental conditions (13, 14). Phosphorylation of GPIbb has previously been shown to inhibit actin polymerization in stimulated platelets (1). However, the effect of GPIbb phosphorylation, and increased 14-3-3x association (15), on von Willebrand factor binding may be more complex than first thought, since separate studies report either up- (13) or down(14) regulation of von Willebrand factor adhesion by either alanine or glycine substitution of serine-166, respectively. Together, this may imply that the conformation of the tail is functionally important, or that these mutations differentially affect other interactions involving the GPIb-IX-V cytoplasmic domain (1; refer below). The major ligand-binding domain of GPIb-IX-V is the Nterminal 282 residues of GPIba. The structure of this domain, based on X-ray crystallography of recombinant GPIba fragments, was published in 2002 (16, 17). The structure reveals a cupped ‘hand’ configuration (16), comprising the leucine-rich repeats and N-terminal flank (fingers and palm), the C-terminal region containing disulphide-bonded residues 209 – 248 and 211 – 264 (thumb), and the anionic sulphated sequence 259 – 282 containing three sulphated tyrosine resi-
dues at 276, 278 and 279 (wrist). The wrist links the globular ligand-binding domain to the membrane-proximal, sialomucin domain in full-length GPIba. The 1 – 282 sequence contains noncontiguous binding sites for the von Willebrand factor A1domain, for a conserved insert (or ‘I’) domain in the aM subunit of the leukocyte integrin, aMb2 (Mac-1), and for Pselectin expressed on activated platelets and endothelial cells (1, 4). This region of GPIba also binds a-thrombin (18, 19), thrombospondin (20), kininogen, and clotting factors XI/XIIa (1). In contrast, collagen binds to GPV (3). Collagendependent aggregation is diminished either by soluble GPV, or in GPV-deficient mouse platelets (that interact normally with von Willebrand factor). The co-crystal structure of a GPIba fragment and a von Willebrand factor A1-domain fragment (both containing gainof-function point mutations to facilitate complex formation) reveals how elements within the N-terminal domain, leucinerich repeats and C-terminal disulphide-knot of GPIba directly interact with A1 (17). Only minor conformational changes of the GPIba fragment accompany binding to von Willebrand factor A1, with the striking exception of the 209 – 248 disulphide-loop. This ‘b-switch’ loop becomes a stabilized bsheet in the complex (17). Congenital gain-of-function point mutations, that enhance von Willebrand factor binding, have previously been pinpointed to this sequence (16, 17). In the complex, a negatively-charged patch encompassing residues 59 – 128 within the leucine-rich repeats of GPIba is opposite to a positively-charged region of von Willebrand factor A1. Electrostatic interactions involving these complementary patches are likely to be functionally important, because cross-species chimeras of full-length, surface-expressed GPIba lacking this negative-charge cluster fail to adhere to von Willebrand factor under hydrodynamic shear flow (4). GPIba can bind a-thrombin in two ways. Independent structural analyses of complexes of a GPIba fragment and athrombin reveal the possibility of two distinct interactions (18, 19). Both structures involve predominantly the C-terminal region of GPIba (disulphide-knot or the anionic/sulphated region). In one structure, thrombin’s catalytic site is accessible, whilst in the other configuration, the sulphated region of GPIba may in part mask a functional site of thrombin. This sulphated sequence of GPIba has been previously implicated as a thrombin-binding site, and is analogous to sulphated sequences in the leech-derived thrombin inhibitor, hirudin, and other thrombin-binding peptides. This infers a potential mechanism for regulation of thrombin activity by GPIba. Previous studies demonstrate GPIba acts as a cofactor for thrombin-dependent activation of the protease-activated receptor, PAR-1, on platelets (1). Recent evidence also supports a direct mechanism for thrombin-induced platelet activation involving binding to GPIba on platelets or where GPV is absent (21 – 23). GPV is also a thrombin substrate (1). GPIb-IX-V transmits intracellular signals in response to von Willebrand factor binding (1, 2). This has several key
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consequences, including elevation of cytosolic Ca2 + , cytoskeletal rearrangements, secretion of ADP, and activation of aIIbb3 (Fig. 1A). Pathways of GPIb-IX-V-dependent signalling have been elucidated, in part, by several recent studies. First, discrete binding sites for 14-3-3x and calmodulin in the cytoplasmic domain of GPIb-IX-V have been defined. As discussed above, 14-3-3x binds to a constitutive phosphorylation site (serine-609) at the C-terminus of GPIba ( . . . SGHpSL), and to a PKA-dependent phosphorylation site (serine-166) of GPIbb ( . . . RLpSLTDP . . . ) (12, 15). Immediately N-terminal to the 14-3-3x-binding site of GPIbb is a positively-charged, membrane-proximal sequence that binds calmodulin (24). A different calmodulin-binding sequence is within the short, *16-residue cytoplasmic tail of GPV. Both 14-3-3x and calmodulin dissociate from GPIb-IXV following platelet activation (15, 24). Reconstitution of GPIb-dependent activation of aIIbb3 in mammalian cell lines has revealed that an intact 14-3-3x-binding site on GPIba is essential for this process, and that activation of ERK-1/2 kinase is a likely downstream mediator (25). It has also been shown that phosphoinositide (PI) 3-kinase is linked to GPIbIX-V, via 14-3-3x, and that src kinase is activated by engagement of GPIb-IX-V (1, 2, 26). Additional studies support a role for Fc receptor g-chain (FcRg) in GPIb-IX-Vdependent signalling, since GPIb-IX-V and FcRg are coprecipitated (27), and GPIb-dependent signals are altered in FcRg-deficient platelets (28). FcgRIIa is also associated with GPIb-IX-V (1). Finally, localization of subpopulations of GPIb-IX-V to cholesterol-rich membrane raft microdomains may also regulate signalling (29). The fraction of total GPIbIX-V in rafts in resting/activated platelets has not been quantified. Complications include variations between laboratories in solubilization and raft-isolating conditions, as well as platelet treatment prior to extraction; the extent to which palmitylation of GPIbb and GPIX regulates raft association, and how palmitylation itself is controlled and maintained in platelets and during analysis, is also thus far less than thoroughly understood.
GPVI GPVI is a major collagen receptor on platelets (3). The role of GPVI in platelet activation and aggregation in the context of other collagen-binding receptors on platelets has been the focus of much recent analysis (see for example, 30 – 33). It is evident, however, that signalling by GPVI is required for optimal platelet aggregation in response to collagen, including upregulation of the collagen-binding integrin, a2b1. Congenital deficiency of GPVI in humans is extremely rare, but abnormal response of platelets to collagen in these patients is associated with only mild bleeding tendency. Similarly, depletion of GPVI from mouse platelets has only minimal affect on bleeding, even though platelets are defective in collagen responsiveness (30, 32). GPVI-deficient platelets in
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Figure 1. (A) Platelet GPIb-IX-V and GPVI. ‘Side-on’ view of GPIba, GPIbb, GPIX, GPV and GPVI. The horizontal black line represents the platelet membrane (extracellular domains, uppermost). Evidence supporting co-association of GPIb-IX-V and GPVI is summarized: (1) Ligands like collagen (GPV and GPVI) and venom proteins (GPIba and GPVI) interact with both receptors; (2) The anti-GPIba monoclonal antibody, SZ2, inhibits collagen-induced platelet aggregation; (3) FcRg has been implicated in signalling responses to both receptors; (4) Cytoplasmic regions of both receptors bind calmodulin; and (5) Engagement of both receptors induces responses such as elevation of cytosolic Ca2+, and ‘inside-out’ activation of aIIbb3. (B) Platelet ‘adhesosome.’ A speculative topographical arrangement for the adhesive subunits of the GPIb-IX-V complex and GPVI, with a GPIb-IX : GPV : GPVI ratio of *4 : 2 : 1. von Willebrand factor, P-selectin and Mac-1 bind GPIba; collagen binds GPV and GPVI; a-thrombin binds GPIba (and cleaves GPV); and venom proteins convulxin, alboaggregin-A and alborhagin bind GPIba and/or GPVI.
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mice may be generated either by depletion of FcRg (required for GPVI surface expression), by antibody-induced shedding of GPVI, or by gene-targeted deletion of GPVI (where FcRg is expressed) (34). Mechanisms of GPVI-dependent platelet activation (Fig. 1A) may be related to three defined interactions of GPVI. First, a membrane-proximal positively-charged consensus motif within the cytoplasmic domain of GPVI binds calmodulin (35, 36). Second, a proline-rich sequence immediately Cterminal of the calmodulin-binding site is a consensus motif for binding SH3 (src homology-3) domains of src-family kinases, and binds to fyn and lyn (36, 37). Third, an arginine residue within the transmembrane domain and additional elements within the cytoplasmic tail mediate the association of GPVI with FcRg (36, 38). Each of these interactions may be independently disrupted in mutated forms of the receptor, allowing dissection of specific functional effects (36 – 38). FcRg-dependent pathways involve receptor cross-linking and autophosphorylation of ITAM (immunoreceptor tyrosinebased activation motif) domains, and activation of syk kinase. Disruption of calmodulin association affects GPVI-induced elevation of cytosolic Ca2 + . Engagement of GPVI can also activate fyn and lyn independent of FcRg. GPVI is capable of binding collagen and a variety of structurally different ligands. These include collagen-related peptides and snake toxins, which recognize partially overlapping, or distinct binding sites. Thus, signalling responses may depend on the nature of the ligand, as well as coincident engagement of one or more additional receptor(s), such as a2b1, GPIb-IX-V or others (2, 3, 30 – 32, 39).
PATHO/PHYSIOLOGY OF GPIb-IX-V AND GPVI GPIb-IX-V and GPVI initiate platelet activation and aggregation. GPIb-IX-V has an established role in mediating platelet adhesion under hydrodynamic shear stress, at higher levels of normal physiological shear, and at pathological levels of shear (1, 2, 4). Consequently, GPIb-IX-V is under vigorous investigation as a target for platelet-based anti-thrombotic therapeutics, notwithstanding the dilemma of selectively maximizing efficacy of GPIb-IX-V blockade in thrombosis, while minimizing potential side effects of bleeding. Plateletbased therapeutic targets, including GPIb-IX-V and other receptors, have recently been reviewed elsewhere (40). Clinical implications of GPIb-IX-V blockade in thrombus formation in vivo is also being elucidated using inhibitory anti-GPIb antibodies in non-human primates (41, 42). Interestingly, recent studies using mouse models of arterial thrombosis show that thrombus development is markedly defective in the absence of GPVI despite normal expression of GPIb-IX-V (43). This supports a dual contribution of GPVI and GPIb-IXV towards formation of a thrombus in this model; as discussed above, however, GPVI deficiency in humans or mice has minimal effect on skin bleeding times, suggesting the precise
role of each receptor depends on the physiological context (for example, arterial vs. peripheral circulation). In addition to a role in thrombosis, it is now apparent that GPIb-IX-V also participates in other vascular processes. First, by interaction with P-selectin, GPIba may mediate adhesion of resting platelets to activated vascular endothelium (1). GPIba can also mediate leukocyte adhesion to adhered platelets by a Mac-1-dependent mechanism (4). Second, GPIba and GPVI promote coagulation by localizing thrombin or other procoagulant factors at thrombotic sites (1, 3, 4). Finally, a recent paper by Hoffmeister and colleagues illustrates the importance of binding of GPIba to Mac-1 on liver phagocytic Kupffer cells in clearance of platelets from the circulation (44). Remarkably, this process is enhanced by low temperature exposure of platelets by a mechanism involving receptor clustering that increases Mac-1-dependent adhesion. This phenomenon explains the low tolerance of platelets to refrigerated storage for the purpose of transfusion (44). Another study by Bergmeier and colleagues using a ‘mitochondrial-injury’ model suggests that decreased GPIba on platelets upon in vitro storage results from a mechanism involving extracellular, metalloproteinase-dependent shedding (45).
CO-ASSOCIATION OF GPIb-IX-V AND GPVI? GPIb-IX-V and GPVI both initiate platelet activation and aIIbb3-dependent aggregation. In view of this parallel physiological function, is it possible that GPIb-IX-V and GPVI are co-associated on the platelet surface? It could be speculated that these receptors may form an adhesive cluster or ‘adhesosome’ (Fig. 1B). Whilst there are, as yet, no definitive data published, several lines of evidence support this possibility: First, GPV and GPVI both bind collagen (3). This would presumably be facilitated if the receptors were proximal. Second, a number of multimeric snake toxins of the C-type lectin family engage both GPIba and GPVI (39). Dual receptor occupancy by these ligands could initiate signalling by cross-linking GPIb-IX-V and GPVI (and thus FcRg). Third, both GPIb-IX-V- and GPVI-mediated signaling may involve FcRg (27, 28, 36, 38). FcRg directly associates with GPVI, supporting its surface expression. Fourth, an antiGPIba monoclonal antibody, SZ2, which maps to the anionic/ sulphated sequence of GPIba inhibits collagen-induced platelet aggregation mediated by GPVI (1). Fifth, both GPIb-IX-V (GPIbb and GPV) and GPVI directly associate via their cytoplasmic domains with calmodulin, interactions that dissociate following platelet activation (24, 35, 36). It is conceivable calmodulin association maintains an adhesive complex on resting platelets. Finally, in addition to upregulation of aIIbb3 activity, transient Ca2 + elevation is a consequence of engagement of both GPIb-IX-V and GPVI (1, 2). In the latter case, this has been linked to the calmodulinrecognition motif of GPVI, a motif conserved in GPIbb (36).
PLATELET INTERACTIONS
It has also been noted that at least subpopulations of both GPIb-IX-V and GPVI are raft-associated in activated platelets (29), and if a GPIb-IX-V-VI complex occurs, then it may be dependent upon the activation state of platelets, and/or may only apply to a fraction of total expressed receptors. In general terms, the arrangement of GPIb : GPV : GPVI of *4 : 2 : 1 shown in Figure 1B would approximate the relative copy number of each receptor (32).
FUTURE RESEARCH Future research will establish the topographical relationship of GPIb-IX-V and GPVI, the functional consequences, if any, of this co-association, and molecular determinants mediating the complex. These lines of investigation will doubtless be extended to consider other platelet adhesion receptors, in particular, a2b1, other collagen-binding adhesion receptors, aIIbb3, and Fc receptors. The result will be a surface map of platelet receptors, and insights into how physical proximity translates into functional efficacy.
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