Lorenzo Alberio, Md Theodor Kocher Institute of the University of Berne, Freiestrasse 1, CH–3012 Berne, Switzerland. Tel.: +41 31 6314148; fax: + 41 31 6313799; e-mail: firstname.lastname@example.org
Platelet adhesion to and activation by exposed subendothelial collagen plays a critical role in normal haemostasis and pathological thrombosis. Recent advances in elucidating the mechanisms underlying platelet–collagen interaction support a ‘two-site, two-step’ model. Direct platelet binding to integrin α2β1 mainly sustains adhesion and allows recognition of glycoprotein VI. The latter interaction is responsible for characteristic intracellular signalling events leading to p72Syk and PLCγ2 activation. The present review describes the known collagen receptors on platelets and discusses the current understanding of signal transduction promoted by collagen.
Platelets play two key roles in haemostasis: upon activation they create a physical barrier that limits blood loss, and they accelerate thrombin generation by providing a surface which catalyzes at least two procoagulant reactions, the conversion of factor X to Xa and of prothrombin to thrombin. These processes are initiated by platelet interactions with the exposed subendothelium at sites of vascular injury [ 1].
At high shear rates the rapid and reversible interaction between glycoprotein (GP) Ib-IX-V and von Willebrand factor (vWF) adherent on collagen types I, III [ 2] and VI [ 3, 4], is crucial for slowing the platelets thereby allowing them to undergo stable binding with other elements of the damaged vessel wall [ 5, 6]. One of the major subendothelial structures that directly reacts with platelets is collagen, which not only offers a substrate for adhesion but also induces platelet activation [ 7, 8]. Indeed, collagen is considered the most thrombogenic matrix component and, together with thrombin, the physiologically strongest platelet agonist [ 9]. Platelet–collagen interactions play therefore a critical role in physiological haemostasis, and understanding this process will help to dissect some events involved in pathological thrombosis. Several reviews have been recently published dealing with different aspects of this interaction [ 7, 10–14].
An emerging model of the direct platelet–collagen interaction supports the ‘two-site, two-step’ hypothesis originally proposed 10 years ago [ 15, 16], based on evidence that integrin α2β1 mediates firm adhesion to collagen, thereby allowing platelet interaction with a lower affinity receptor, GPVI, mainly responsible for activation ( Figure 1). In the present work we will describe the known collagen receptors on platelets and discuss the current understanding of intracellular signalling promoted by collagen. This review does not cover the important search for platelet-reactive structures of the collagen molecule [ 17, 18].
Collagen receptors on platelets
Over the years many studies have identified potential platelet receptors for collagen. This is a difficult interaction to dissect because: of the 18 described collagen types at least 7 are present in the vessel wall [ 19]; collagen is an insoluble macromolecular protein at physiologic pH making analysis of cell–protein interactions difficult [ 20]; and many activation end-points, including membrane GP trafficking, granule secretion and aggregation, progress simultaneously when platelets bind collagen [ 12]. In spite of these obstacles, several proteins on the platelet surface, both integrin and nonintegrin, have been shown to possess characteristics consistent with being specific collagen receptors.
Integrin α2β1 (glycoprotein Ia-IIa)
Integrin α2β1, which is identical with the platelet membrane GP Ia-IIa, the very late activation antigen-2 on activated T cells [ 21] and the class II extracellular matrix receptor on fibroblasts [ 22], has recently been reviewed in depth [ 23]. The α2 (GPIa) and β1 (GPIIa) subunits have apparent molecular weights of 165 kDa (reduced) and 130 kDa, respectively. The aminoacid (aa) sequences were deduced from their corresponding cDNA [ 24, 25]. Both subunits possess a short C-terminal cytoplasmic tail, a single hydrophobic membrane spanning region and a large extracellular domain. The α2 chain, like most other integrin α subunits, does not undergo post-translational cleavage. Its extracellular domain contains a 7-fold repeat segment which includes a EF hand motif with three cation binding sites thought to be involved in ligand binding [ 26]. Just distal to these, there is a segment of 191 aa called the I (inserted)-domain, which is homologous to the collagen-binding A domain of vWF [ 24]. In fact, the α2 I-domain has been shown to bind collagen [ 27, 28], and a recombinant α2 I-domain fusion protein inhibits collagen-induced platelet adhesion [ 29]. While the α2 I-domain is sufficient and essential for platelet-collagen binding, other structures, such as the adjacent EF hand motif can optimize it [ 26]. In addition, the extracellular domain of the β1 subunit, which is composed of a proximal portion of four internally folded cysteine-rich repeat units and a distal portion of highly conserved sequence shared with other β integrins, is thought to regulate the binding affinity of the α2 I-domain [ 30, 31].
The first indication that integrin α2β1 might be a physiologically relevant collagen receptor came from the observation of a female patient with excessive post-traumatic bleeding and menorrhagia [ 32]. This patient's platelets, which selectively failed to aggregate or undergo shape change in response to collagen, were found to contain only 15–20% of the normal amount of GPIa [ 32]. Furthermore, under flow conditions these platelets exhibited a markedly decreased adhesion to and failure to spread on a subendothelial surface [ 33]. A second patient with GPIa deficiency has been reported, whose haemorrhagic symptoms and biochemical defect surprisingly disappeared when she entered menopause [ 34]. In addition, a patient with an acquired bleeding disorder associated with an autoantibody against GPIa [ 35] and another with a complete deficiency of GPIa in the context of a myeloproliferative disorder [ 36] have been described.
The interaction of integrin α2β1 with collagen requires Mg2+ (which can be substituted by Mn2+, Co2+, Cu2+, Fe2+ and Zn2+) and is inhibited by Ca2+ [ 37, 38], thus explaining the observation that platelet adhesion to collagen is markedly increased in the presence of Mg2+ [ 39]. Saelman et al. demonstrated that collagens I through VIII are variously able to support platelet adhesion and that this can be completely inhibited, under conditions of both stasis and flow, by a monoclonal antibody directed against GPIa [ 40]. Noteworthy, collagen type V does not support adhesion under flow, and at shear rates of 1600/s adhesion to collagen type III is only inhibited by 85% [ 40]. Interestingly, at high shear rates inhibition of platelet adhesion to the most reactive collagens, types I through IV, requires three times more antibody than needed at low shear [ 40], suggesting that the role of integrin α2β1 is more critical at high shear. Consistent with this are studies showing that the binding avidity of integrin α2β1 can be modulated with antibodies against the β1 subunit [ 30, 31], and enhanced following activation of protein kinase C [ 41]. Studies with soluble collagen indicate that integrin α2β1 becomes activated following platelet interaction with thrombin, ADP and a GPVI specific agonist [ 42]. Finally, CD47/IAP (integrin-associated protein), a receptor for the cell binding domain of thrombospondin-1, has recently been shown to coimmunoprecipitate with integrin α2β1 and to augment its function [ 43]. These data suggest the interesting hypothesis that variations in integrin α2β1 binding avidity may have physiological significance.
The number of integrin α2β1 molecules on the platelet surface significantly varies between normal individuals [ 44], ranging between 800 and 1800 [ 21, 44, 45]. While platelet activation does not increase the number of surface molecules by more than 5% [ 44], the interindividual variation of integrin α2β1 surface levels by itself correlates with platelets' ability to adhere to collagen types I and III under static conditions [ 44] and to collagen type I under flow [ 46]. This heterogeneity is associated with three α2 gene alleles, defined by eight nucleotide polymorphisms [ 46, 47]. Initially two silent, linked polymorphisms located at nucleotides 807 (TTT/TTC at codon Phe224) and 873 (ACA/ACG at codon Thr246) were described [ 47]. Although the aa sequence of the α2 subunit is not affected by the polymorphisms, the 807T/873A pair is associated with higher surface levels of integrin α2β1 than the 807C/873G pair [ 47]. Subsequently, a similarly silent but much rarer polymorphism located at nucleotide 837 (C or T) and linked to the Br polymorphism [ 48] was also identified [ 46]. Allele 1 (807T/837T/873A/Brb) has a frequency of ≈ 39% and is associated with increased levels of integrin α2β1, while allele 2 (807C/837T/873G/Brb) with a frequency of ≈ 53% and allele 3 (807C/837C/873G/Bra) with a frequency of ≈ 8% are both associated with lower levels of integrin α2β1 [ 46]. In a case control study, a significantly higher prevalence of individuals homozygous for 807T/873A were found among patients with myocardial infarction [ 49], and multivariate analysis confirmed this genotype as an independent risk factor for myocardial infarction [ 49]. Recently, the 807T allele was found to strongly correlate with the development of nonfatal myocardial infarction [ 50] and stroke [ 51] in younger patients. These three studies indicate that the inherited variation of platelet surface integrin α2β1 is a significant risk factor for acute thrombotic events. Interestingly, the frequency of the 807C allele, which is associated with a lower integrin α2β1 density and a decreased haemostatic function, has been shown to be significantly higher among type 1 von Willebrand disease patients than among normal individuals [ 52]. This observation confirms that integrin α2β1 is a significant contributor to the haemostatic process, and suggests that its surface variability among patients with similar vWF levels may account for the very different bleeding phenotypes.
In summary, integrin α2β1 is considered to be the major receptor mediating direct and permanent platelet adhesion to collagen [ 11], thus perfecting the initial vWF-dependent tethering at high shear rates [ 6] and facilitating engagement of lower affinity receptors, such as GPVI. The extent of integrin α2β1 contribution to subsequent platelet activation is less clear (see below). The clinical relevance of integrin α2β1 polymorphisms with its variable expression on the platelet surface has been addressed in a recent review [ 53].
Glycoprotein VI (p62)
GPVI, a not yet cloned 62 kDa (reduced) platelet membrane protein, was first described 20 years ago [ 54], and its involvement in platelet–collagen interactions was postulated one decade later based on the following observations. An antibody that recognized a 62/57 kDa platelet membrane protein was identified in the serum of a patient with autoimmune thrombocytopenia whose platelets were selectively defective in collagen-induced aggregation [ 55]. This antibody could recognize a 62 kDa protein and induce aggregation of normal platelets [ 55], but did not react with platelets from a GPVI-deficient patient [ 56], thus identifying the 62 kDa protein as GPVI and demonstrating its involvement in collagen-induced platelet activation. To date, 3 GPVI-deficient patients [ 56–58], and two patients with an autoantibody against GPVI [ 55, 59], have been described. All patients exhibit a mild bleeding tendency and slightly prolonged bleeding times. While their platelets have an essentially normal response to other physiologic agonists, they show a defective aggregation in response to collagen despite normal expression of integrin α2β1. Although GPVI has been implicated in platelet adhesion to collagen under static conditions [ 58, 60, 61], this is probably a consequence of its ability to induce platelet activation. Under flow conditions GPVI involvement relates to second phase adhesion, a process which is secondary to integrin αIIbβ3 activation [ 62]. GPVI has been demonstrated to recognize both the tertiary (triple-helical) and quaternary (polymeric) structure of collagen and is considered to be the crucial receptor mediating platelet activation [ 63]. The intracellular signalling events induced by GPVI engagement will be discussed below.
CD 36 [ 64], an 88 kDa GP functioning as a scavenger receptor and cell-adhesion molecule [ 65], is expressed on several cell types, including platelets, monocytes/macrophages, reticulocytes/erythrocytes, microvascular endothelial cells and melanoma cells, and has been implicated in a variety of pathophysiological situations ranging from haemostasis and thrombosis to malaria, inflammation, lipid metabolism and atherogenesis [ 65].
It has been estimated that there are about 20 000 CD36 molecules on the platelet surface [ 66]. This GP has been proposed as a collagen receptor based on the observation that antibodies against it could inhibit collagen induced platelet activation and aggregation [ 67, 68]. In addition, incubation of normal platelets with Fab fragments of a monospecific polyclonal anti-CD36 antibody inhibited the early stages of adhesion to collagen type I under static [ 67] and under flow conditions [ 69]. Diaz-Ricart et al. [ 69] using citrated reconstituted whole blood also showed that platelets from CD36-deficient individuals have a decreased early adhesion. However, 3% to 11% of healthy Japanese blood donors lack CD36 without any apparent bleeding disorder [ 70]. Moreover, collagen induced aggregation [ 71] and metabolic responses [ 72] in CD36-deficient platelets have been shown to be normal. The discrepancies between these observations and the previous studies might reside in the divalent cation conditions employed. Utilyzing heparinized blood, Saelman et al. demonstrated that CD36-deficient platelets adhere normally to collagen type I, III, and IV under both static and flow conditions [ 73]. Remarkably, while collagen type V is not adhesive during flow [ 40], under static conditions adhesion of both homozygous and heterozygous CD36 deficient platelets to this collagen type was strongly reduced [ 73]. The peculiar behaviour of collagen type V was confirmed by Kehrel et al. who showed that CD36-deficient platelets aggregate normally with collagen types I and III but not in response to collagen type V [ 74]. Indeed, CD36-deficient platelets appeared even more sensitive to types I and III collagens than normal platelets [ 74], suggesting an inhibitory co-operation between CD36 and other collagen receptor(s). Taken together these observations seem compatible with the hypothesis that CD36 might be involved in the very first adhesion of platelets to collagen, but it is essential only for interaction with collagen type V. This may be relevant to the development of thrombotic events because collagen type V is increased in atherosclerotic plaques [ 75].
Chiang and Kang have isolated and purified from human platelets a 65 kDa protein which behaves as a receptor for collagen type I [ 76]. They subsequently demonstrated that platelet aggregation induced by collagen type I can be inhibited with poly- and monoclonal antibodies against the 65 kDa protein [ 77, 78]. Finally, they succeeded in cloning a cDNA strand that encodes the 65 kDa receptor, showing that the recombinant protein binds to and inhibits platelet aggregation and ATP release in response to collagen type I [ 79]. They also have identified a portion of the receptor molecule likely to represent the collagen binding site [ 80]. Interestingly enough, this body of work demonstrates that the 65 kDa protein is not involved in the platelet interaction with collagen type III.
Deckmyn et al. described a patient with an antibody directed against a 85/90 kDa platelet membrane GP which interfered with collagen-induced platelet aggregation [ 81]. Despite similar electrophoretic behaviour, purified CD36 was not recognized by the patient's antibody indicating that the 85/90 kDa GP is a distinct structure [ 81]. Little else is known about this putative collagen receptor.
Signal transduction induced by collagen
One set of early events in platelet activation induced by most agonists involves the hydrolysis of inositol phospholipids [ 82]. Following receptor engagement, activated phospholipase C (PLC) cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to the second messengers inositol 1,4,5-trisphosphate (IP3) and 1,2-diacyglycerol (DAG). IP3 increases the cytosolic Ca2+ concentration by releasing stored Ca2+ from the dense tubular system and DAG activates protein kinase C. These events promote thromboxane A2 (TxA2) generation, shape change, granule secretion, activation of the fibrinogen receptor integrin αIIbβ3 (glycoprotein IIb-IIIa) and ultimately platelet aggregation [ 82].
Platelets contain at least two forms of PLC, β and γ, which are regulated through different mechanisms involving heterotrimeric G-proteins and protein tyrosine kinases (PTK), respectively [ 83]. Accordingly, thrombin and TxA2, whose platelet receptors are known to signal by means of G proteins, have been shown to activate PLCβ. As for collagen, it induces a PTK-dependent mechanism for phosphorylation of PLCγ2, the predominant isoform of PLCγ in platelets, which is not activated following stimulation with thrombin or the stable TxA2 analogue U46619 [ 84, 85]. These observations confirm that the two strongest physiological platelet agonists, thrombin and collagen, operate through different signalling pathways [ 86].
Collagen promotes tyrosine phosphorylation of numerous proteins in platelets [ 87]. While the phosphorylation of several of them depends on integrin αIIbβ3 engagement and platelet aggregation, at least two proteins of 38 and 72 kDa, respectively, are rapidly and markedly phosphorylated even when events downstream of PLC are selectively inhibited [ 88], suggesting their involvement in early signalling between collagen receptor engagement and PLCγ2 activation. The 72 kDa protein has been identified as Syk [ 89] and the 38 kDa protein is likely to be the human equivalent [ 90] of LAT (linker for activation of T cells), a recently cloned adapter protein implicated in T-cell receptor signalling [ 91]. Furthermore, the 38 kDa protein, Syk and PLCγ2 also become phosphorylated following cross-linking of the low affinity receptor for IgG complexes present on platelets, FcγRIIA (CD 32), suggesting that collagen-induced platelet activation might be mediated through the same pathway as that used by immune-receptors [ 88].
Collagen signals by a similar pathway as immune receptors
Immune receptors operate through sequential activation of members of the Src and Syk kinase families with a pivotal role played by a tyrosine-based motif located in the cytoplasmic tail of the receptor itself or its associated chain [ 92]. This motif, identified in 1989 and now known as the immunoreceptor tyrosine-based activation motif (ITAM), is defined by the sequence YXXL/IX(6–8)YXXL/I, where X represents any amino acid [ 92]. Upon receptor activation, the ITAM becomes phosphorylated on both conserved tyrosine residues by a member of the Src family. This allows association with the tandem Src homology 2 (SH2) domains of Syk, leading to its activation and downstream signalling, including PLCγ2 phosphorylation.
In order to identify ITAM-containing proteins that undergo phosphorylation following collagen stimulation, Gibbins et al. have incubated platelet lysates with the tandem SH2 domains of Syk expressed as a glutathione-S-transferase fusion protein [ 93]. They found that the Syk tandem SH2 domains precipitated a tyrosine phosphorylated protein with a molecular weight of 12/25 kDa (reduced/nonreduced), which was identified as the Fc receptor (FcR) γ-chain [ 93]. The FcR γ-chain is expressed in cells as a homodimer linked by a disulfide bridge. It plays a role in surface expression and function of the receptor for IgA (FcαR), the high affinity receptors for IgE (FcεRI) and IgG (FcγRI), and the low affinity IgG receptor (FcγRIII) [ 94–97]. It should be noted that FcγRIIA, the low affinity receptor for immune complexes expressed on platelets, does not associate with the FcR γ-chain but contains an ITAM sequence on its own cytoplasmic tail. This immune receptor is not directly involved in collagen signalling because activation of the ITAM sequence by receptor clustering does not induce phosphorylation of the FcR γ-chain. The concept that collagen activates platelets through a pathway involving tyrosine phosphorylation of FcR γ-chain, Syk and PLCγ2, a signalling sequence characteristic of immune receptors, was confirmed in knock-out mice lacking either the FcR γ-chain or Syk [ 98].
GPVI and FcR γ-chain constitute a collagen receptor complex
Studies performed with collagen-like, triple helix peptides based on a glycine-proline-hydroxyproline repeat sequence, which cannot bind to integrin α2β1, indicated that platelet activation and aggregation [ 99], and tyrosine phosphorylation and activation of Syk and PLCγ2 [ 100], can be achieved through a different collagen receptor. This was supported by Ichinohe et al. [ 101], who observed that GPVI cross-linking induced platelet activation in a manner similar to collagen: it is not inhibited by elevation of intracellular c-AMP [ 86], and promotes a PTK-dependent activation of c-Src, Syk, and PLCγ2 [ 102, 103]. Furthermore, the same group reported that GPVI-deficient platelets expressing normal amounts of α2β1 exhibited a defect in tyrosine phosphorylation of Syk, Fak, PLCγ2 and Vav [ 104], while activation of c-Src did occur and could be abolished by a mAb against α2β1 [ 104]. The final link between GPVI and the collagen signalling pathway previously described was established by demonstrating that GPVI and the FcR γ-chain are both expressed as a functional unit on the surface of normal platelets and proportionally decreased in GPVI-deficient platelets [ 105, 106]. This concept was independently confirmed by Polgár et al. who demonstrated that convulxin, a powerful platelet activator isolated from the venom of Crotalus durissus terrificus, selectively binds to GPVI inducing a signal transduction like collagen, and that convulxin subunits are able to inhibit both platelet aggregation and tyrosine phosphorylation in response to collagen [ 107].
Recent reports are further clarifying the early signalling events initiated by GPVI cross-linking. First, Ezumi et al. employing Sepharose 4B beads coupled with the specific GPVI agonist convulxin [ 107] for affinity precipitation of the collagen receptor, showed that the Src family PTKs Fyn and Lyn are constitutively associated with the GPVI/FcR γ-chain complex [ 108]. Fyn becomes rapidly phosphorylated upon collagen stimulation and the selective Src family inhibitor PP1 (4-amino-5-[4-methy1pheny1]-7-[t-buty1]pyrazo1 o[3,4-d]pyrimidine [ 109]) inhibits in a concentration-dependent manner tyrosine phosphorylation of FcR γ-chain, Syk, and PLCγ2, granule release reaction, and aggregation [ 108, 110]. This finding demonstrated that Fyn and Lyn are functionally relevant for collagen induced platelet activation. Moreover, the inhibition of FcR γ-chain and Syk phosphorylation by PP1 suggest that either Fyn, Lyn, or both, play a major role in early signalling. Briddon and Watson have suggested that Fyn is constitutively associated either directly with the FcR γ-chain or with another component of the collagen receptor, and that Lyn is involved downstream of the FcR γ-chain because, contrary to Fyn, it associates with several other tyrosine-phosphorylated proteins, including PLCγ2, in a much larger signalling complex [ 110]. Second, Clements et al. have demonstrated that aggregation and tyrosine phosphorylation of PLCγ2 is absent in SLP-76 deficient platelets [ 111]. SLP-76 (Src homology 2 domain-containing leukocyte protein of 76 kDa) is believed to be an essential adapter protein in T cells: it becomes tyrosine-phosphorylated upon T-cell receptor stimulation [ 112], is necessary for tyrosine phosphorylation of PLCγ1 and activation of the Ras pathway in Jurkat cells [ 113], and is required for normal thymocyte development in mice [ 114, 115]. As collagen signals by a pathway similar to immune receptors, it is no surprise that SLP-76 appears to be a crucial adapter protein in collagen-stimulated platelets as well [ 111]. Here it provides, together with downstream SLAP-130 (SLP-76-associated phosphoprotein of 130 kDa), an important link between Syk activation and PLCγ2 regulation [ 116].
In summary, although the signalling pathway downstream of GPVI engagement is not yet completely elucidated, some of its components have been accurately described ( Fig. 1). According to the current model, GPVI is noncovalently associated with FcR γ-chain and with at least one member of the Src family of tyrosine kinases, Fyn. Upon GPVI clustering, the conserved tyrosine residues on the FcR γ-chain ITAM become phosphorylated, presumably by Fyn. This promotes recruitment to the receptor complex of tyrosine kinase Syk, mediated by the association of its tandem SH2 domain with the phosphorylated FcR γ-chain. The activation of Syk, by a mechanism that appears to involve Lyn, leads by means of SLP-76 to tyrosine phosphorylation and activation of PLCγ2 and to the subsequent formation of the two important second messengers, IP3 and DAG.
The interesting hypothesis that signalling generated by the GPVI/FcR γ-chain complex might diverge into a second pathway involving phosphatidylinositol (PI) 3-kinase has been recently proposed by Gibbins et al. [ 90]. PI 3-kinase mediates inositol phospholipid metabolism by converting phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3), a second messenger involved in membrane binding of proteins with pleckstrin homology domains and in the regulation of some protein kinase C isoforms [ 117]. Platelets contain at least one isoform of PI 3-kinase, consisting of a p110 subunit whose subcellular localisation and catalytic activity are regulated by a p85 subunit [ 117]. Gibbins et al. have demonstrated that the p85 regulatory subunit is able to bind both to the tyrosine phosphorylated ITAM sequence of the FcR γ-chain and LAT, the above mentioned 38 kDa protein which becomes phosphorylated in collagen and convulxin stimulated platelets [ 90], thereby providing the potential for a second signalling pathway initiated by GPVI engagement.
The role of other collagen receptors
While the described body of work represents strong evidence for a primary role played by GPVI in collagen induced signalling, relatively little is known about the contribution of other collagen receptors. GPVI-deficient platelets still manifest activation of Src and tyrosine phosphorylation of cortactin in response to collagen, which can be eliminated with an inhibitory mAb against α2β1 [ 104], suggesting that engagement of this integrin promotes intracellular signalling. Consistent with this proposal, collagen induced tyrosine phosphorylation of Syk and PLCγ2 in normal platelets is compromised by blocking integrin α2β1 [ 118] or by proteolytic cleavage of the β1 subunit [ 119]. Taken together, these results indicate that integrin α2β1 may sustain a co-operative role in collagen induced activation of PTKs. However, direct crosslinking of α2β1 with mAbs induces an increase in tyrosine phosphorylation of Syk and PLCγ2 which is dependent on costimulation of FcγRIIA, because specific F(ab′)2 fragments are ineffective [ 118]. The role of FcγRIIA is not yet clarified and this receptor is not specific to collagen signalling. In fact, antibodies directed against other antigens on the platelet surface, such as CD 36 (see below), heparin complexed to PF4 [ 120] or vWF bound to GPIb [ 121] lead to platelet activation through the FcγRIIA receptor.
CD36 is physically associated with the Src family PTKs Fyn, Yes and Lyn in human platelets [ 122], and, similarly to integrin α2β1, mAbs against CD36 can induce platelet secretion and aggregation in a FcγRIIA-dependent manner [ 123], implying its involvement in intracellular signalling as well. However, CD36 appears to be essential only for platelet interaction with collagen type V [ 74]. Finally, a role for the collagen type I specific receptor p65 is suggested by the fact that an anti-p65 mAb inhibits collagen-induced platelet aggregation [ 78]. In summary, besides GPVI other collagen receptors also appear to mediate intracellular signalling; however, their relative contributions and relevance with respect to different collagen types is unclear.
Evidence that each collagen type utilyzes a unique set of platelet receptors
Several observations indicate that each collagen type might interact with platelets through a specific set of receptors. For example, platelet adhesion to collagen type III cannot be completely inhibited by monoclonal antibodies against integrin α2β1 [ 40], and surface level of integrin α2β1 correlates with the lag time before onset of aggregation induced by collagen type I but not by type III [ 44]. Collagen type IV is among the strongest inducers of platelet adhesion [ 40] but does not promote platelet aggregation [ 124] nor activation [ 8]. As already discussed, the 65 kDa receptor studied by Chiang and Kang is specific for collagen type I [ 125] and CD36 is critical only for collagen type V [ 79]. Moreover, most of the studies delineating collagen induced intracellular signalling have been performed with specific agonists for GPVI or collagen type I, so that presently it is not known whether other collagen types might activate different pathways. The existence of diverse, possibly collagen-type specific, mechanisms of collagen–platelet interactions and the relative contributions of the several identified receptors needs further study.
The use of receptor specific agonists, such as aggretin for integrin α2β1 [ 126], convulxin for GPVI [ 107] and collagen-like peptides, which have recently been shown to be specific for GPVI [ 127, 128], together with studies employing receptor specific inhibitory antibodies in platelets simulated with different collagen types should provide some insight into this complex and important area of research.
Note in proof
The collagen receptor GPVI has now been cloned and its sequence published [ 129].
We are indebted to Kenneth J. Clemetson, PhD, for his careful review of the manuscript and helpful suggestions. L. Alberio was supported by a grant from the Swiss National Science Foundation. Additional support was provided by the W. K. Warren Medical Research Institute, and grant HL53585 from the National Institute of Health.