• collagen;
  • GPVI;
  • integrin;
  • Src kinases;
  • Syk;
  • tyrosine kinases


  1. Top of page
  2. Abstract
  3. Introduction
  4. GPVI signaling
  5. Integrin signaling
  6. Signalling by other platelet integrins
  7. Thrombus formation in vitro and in vivo
  8. Conclusions
  9. Acknowledgements
  10. References

Summary.  This review summarizes recent developments in our understanding of the molecular basis of platelet activation by two distinct types of surface receptor, the immunoglobulin GPVI, and the integrin αIIbβ3 (also known as GPIIbIIIa). These two classes of receptor signal through similar yet distinct tyrosine kinase-based signaling cascades leading to activation of phospholipase C γ2. The significance of these signaling cascades in platelet adhesion and platelet aggregation at arterial rates of shear is discussed.


  1. Top of page
  2. Abstract
  3. Introduction
  4. GPVI signaling
  5. Integrin signaling
  6. Signalling by other platelet integrins
  7. Thrombus formation in vitro and in vivo
  8. Conclusions
  9. Acknowledgements
  10. References

The last few years have seen substantial progress in our understanding of the molecular events that underlie activation of platelets by surface receptors for adhesion molecules, most notably those for collagen, fibrinogen and von Willebrand factor (VWF). Much of this progress is due to studies on murine platelets that are deficient in one or more signaling proteins. This research has led to the realization that different classes of adhesion receptors signal through tyrosine kinase-based signaling cascades that utilize many of the same proteins, but which are fundamentally distinct. In this brief review, we shall discuss recent developments in our understanding of the signaling cascades used by the major signaling receptor for collagen, the immunoglobulin (Ig) family receptor, GPVI, and by the major platelet integrin, αIIbβ3. The role of signaling by these two receptors in supporting platelet adhesion and aggregation under flow is considered.

GPVI signaling

  1. Top of page
  2. Abstract
  3. Introduction
  4. GPVI signaling
  5. Integrin signaling
  6. Signalling by other platelet integrins
  7. Thrombus formation in vitro and in vivo
  8. Conclusions
  9. Acknowledgements
  10. References

GPVI is the major signaling receptor for collagen on the platelet surface [1,2]. It has two Ig domains, a mucin-rich stalk and a cytosolic sequence of 51 and 27 amino acids in human and mouse, respectively (Fig. 1). It is coupled to a disulfide-linked Fc receptor (FcR) γ-chain homodimer in the membrane via a salt-bridge between charged amino acids within the transmembrane sequences and through specific sequences with the cytosolic tails [3]. The FcR γ-chain is essential for expression of GPVI on platelets, although interestingly not so in a number of cell lines. The GPVI cytosolic tail contains recognized sequence motifs for binding to calmodulin and the SH3 domain of Src family tyrosine kinases [4–6]. Each FcR γ-chain contains one copy of an immunoreceptor, tyrosine-based activation motif (ITAM) that undergoes phosphorylation on two conserved tyrosines upon crosslinking of GPVI, leading to binding and activation of the tyrosine kinase Syk, and initiation of downstream signaling events.


Figure 1. Structural features of GPVI. GPVI has two extracellular immunoglobulin domains, which are held away from the cell surface by a mucin-like stalk that is rich in o-glycosylation sites. The orientation of the two Ig domains is based on a previous model [1]. There is one site of N-glycosylation in the Ig domain. The GPVI tail is divided into four regions as shown. The proline-rich region recruits Src kinases to the membrane which mediate phosphorylation of the ITAM in the FcR γ-chain (FcRγ). Binding to FcRγ is mediated by a salt-bridge bond in the transmembrane domains of the two receptors and the basic region of the GPVI tail.

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In addition to GPVI, collagen also binds to integrin α2β1 and possibly to other surface receptors. Because of the potential of these receptors to signal, much of our understanding of GPVI signaling is based on studies using GPVI-specific ligands. These include collagen-related peptides (CRPs) that contain the specific GPVI recognition motif, GPO, in a helical confirmation; GPVI-specific antibodies, crosslinked via secondary antibodies; and the snake venom toxin convulxin. Convulxin is the most powerful of these stimuli, most likely because it is tetrameric [7] and therefore capable of crosslinking four GPVI receptors. However, it also binds to GPIb-IX-V [8], although this is not required for functional activation of GPVI as demonstrated using transfected cell line models.

The analysis of the action of these stimuli on human and genetically modified mouse platelets have established many of the key features of the GPVI signaling cascade. Crosslinking of GPVI induces Src kinase-dependent tyrosine phosphorylation of the FcR γ-chain ITAM leading to recruitment and activation of the tyrosine kinase Syk. The two Src family kinases, Fyn and Lyn, play critical roles in this initial step [9], although at least one additional member of the Src family is also involved, as a residual phosphorylation of FcR γ-chain is observed in mouse platelets deficient in Fyn and Lyn, in contrast to the complete abolition of phosphorylation mediated by an inhibitor of Src family kinases [10]. Fyn and Lyn are constitutively bound to the proline-rich region of the GPVI cytosolic tail and crosslinking of the Ig receptor is proposed to bring the two kinases into contact with their substrate, the FcR γ-chain ITAM [5,6]. This is not the only mechanism of ITAM phosphorylation, however, as deletion of the proline-rich binding domain strongly impairs but does not abolish signaling through GPVI [3]. The molecular regulation of this second route of phosphorylation and its dependency on Fyn and Lyn is not known.

Syk undergoes autophosphorylation and phosphorylation by Src kinases upon binding to the phosphorylated ITAM. Syk then initiates a downstream signaling cascade that has many similarities with that used by immune receptors, such as the T- and B-cell receptors on lymphocytes. Central to this signaling cascade is the formation of a signalosome that is composed of a series of adapter and effector proteins. Adapters form an intracellular scaffold that regulates and targets effector proteins to appropriate regions of the cell, thereby bringing them into contact with their molecular substrates. At the core of this signalosome is the transmembrane adapter LAT and the two cytosolic adapters SLP-76 and Gads (Fig. 2; for review see ref. [11]). These three proteins associate with a number of signaling molecules to regulate one of the major effector enzymes in the GPVI signaling cascade, phospholipase C (PLC)γ2, which liberates the second messengers 1,2-diacylglycerol and inositol 1,4,5-trisphosphate. Interestingly, the contribution of the three adapters to the regulation of PLCγ2 is quantitatively distinct. Activation of PLCγ2 by GPVI is almost entirely abolished in the absence of SLP-76, whereas it is strongly or mildly impaired in the absence of LAT or Gads, respectively [12]. Furthermore, the degree of activation of PLCγ2 by GPVI in the absence of LAT is sufficient to elicit a full aggregation response in platelets in response to collagen and GPVI receptor ligands, thereby unmasking a functionally relevant, LAT-independent route of platelet activation. In comparison, LAT plays a non-redundant role in the regulation of PLCγ1 downstream of the T-cell receptor [12].


Figure 2. GPVI signaling cascade. Crosslinking of GPVI induces tyrosine phosphorylation of the FcR γ-chain ITAM by the Src kinases, Fyn and Lyn, which are constitutively bound to the proline-rich region in the GPVI cytosolic tail. This initiates a Syk-dependent signaling cascade that leads to formation of an LAT signalosome and activation of PLCγ2. PLCγ2 associates directly with LAT, and indirectly via the adapters Gads and SLP-76. PLCγ2 also associates with the membrane via binding of its PH domain to PIP3. Functional homologues from the Tec and Vav families support activation of PLCγ2.

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The difference in the role of LAT in the GPVI- and T-cell signaling cascades raises the question as to whether there is a LAT-like transmembrane adapter in platelets that is able to support the GPVI signaling cascade. Several new transmembrane adapters have been identified over the course of the last few years, primarily through data-mining of the human genome. Many of these have been shown to be expressed in hematopoietic cells. These include the membrane adapter NTAL (also known as LAB) which has a similar structure and chromosomal organization as LAT. NTAL also has a short extracellular tail and nine tyrosine residues, many of which are believed to undergo phosphorylation upon crosslinking of ITAM receptors [13]. Interestingly, none of the tyrosines in NTAL fall in the optimal sequence for binding to one of the two SH2 domains in PLCγ1 or PLCγ2, although NTAL does have several potential phosphorylation sites for association with the SH2 domain of Gads, which indirectly regulates PLCγ via association with SLP-76, which itself binds to the P-domain in PLCγ [14]. In contrast, LAT contains tyrosines in optimal sequences for binding to PLCγ and to Gads, and both of these sequences have been shown to be capable of independently binding to PLCγ and supporting activation [15]. Thus, NTAL has the potential to regulate PLCγ2 through one of the two routes used by LAT and is therefore a candidate for mediating LAT-independent regulation of PLCγ2 in platelets as has been demonstrated in mast cells [16,17]. Surprisingly, however, studies on NTAL and NTAL/LAT knockout mice have failed to establish a role for the adapter in the regulation of PLCγ2 by GPVI in platelets, despite the fact that it undergoes marked tyrosine phosphorylation upon activation of the Ig receptor (A.C.Pearce and P.Draber, unpublished data). A probable explanation for this is that NTAL is not phosphorylated in platelets on the tyrosine that mediates binding to Gads. It is not known whether platelets express other transmembrane adapter proteins that could support activation of PLCγ2 by GPVI.

An apparent difference between the signaling cascades used by GPVI and other ITAM receptors that has emerged in recent years is in regard to the role of the Vav family of GDP/GTP exchange factors (GEFs). Studies using mice deficient in one or more of this family have demonstrated a critical role for Vav in supporting cell activation by ITAM receptors, including the T-cell antigen receptor, which also signals through the LAT-Gads-SLP-76 signalosome (for review see Ref. [18]). The role of Vav in supporting the activation of PLCγ cascade is likely to be a consequence of its role as an adapter protein [19], supported by its multidomain structure which includes SH2 and SH3 domains. There is no direct evidence to posit a role for the GDP/GTP activity of Vav in supporting the activation of PLCγ. Studies on platelets from mice deficient in Vav1, Vav2, or in Vav1/Vav2, however, did not reveal a defect in PLCγ2 activation by GPVI, despite robust tyrosine phosphorylation of Vav1 in control platelets [20]. On the other hand, Vav2, which is expressed at low level, does not undergo tyrosine phosphorylation. Interestingly, the remaining member of this family, Vav3, has been recently shown to be expressed in platelets [21] and to undergo tyrosine phosphorylation in response to activation of GPVI [22]. Mice deficient in Vav3 however also do not exhibit an impairment in activation in response to stimulation of GPVI, despite the fact that there are no apparent compensatory changes in the levels of expression of the other Vav family proteins (and vice versa in Vav1 and Vav2 knockout platelets) [22]. The molecular explanation for this apparent discrepancy with regard to the role of Vav family proteins was revealed through studies on platelets deficient in both Vav1 and Vav3. Platelets deficient in these two members of the Vav family exhibit a dramatic reduction in tyrosine phosphorylation of PLCγ2 and associated functional responses downstream of GPVI [22]. A similar set of results is seen in the absence of all three members of the Vav family of proteins, demonstrating that the weak residual activation response is independent of Vav. Thus, this emphasizes the conservation of the fundamental features of ITAM-based signaling cascades in hematopoietic cells, namely that all are dependent to a very large extent on Vav family proteins.

The studies on Vav demonstrate an emerging paradigm, namely that many of the proteins in the GPVI signaling cascade have one or more functional homologs that share overlapping functions. However, in contrast to the situation with Vav, deletion of any of the functional homologues is usually sufficient to generate a phenotype, with one of the proteins usually having a much greater role than the others in supporting activation. Examples of proteins with functional homologs in the GPVI signaling cascade are discussed below.

Tec kinases are the third family of tyrosine kinases that regulate PLCγ downstream of ITAM-based signaling cascades. A role for the Tec family kinase Btk in signaling downstream of the B-cell antigen receptor was discovered through studies on patients with the immunodeficiency X-linked agammaglobulinaemia (XLA). This disorder is caused by a functional defect in Btk, leading to a dramatic impairment in activation of PLCγ1 by the B-cell receptor during B-cell development and a resultant deficiency of mature B lymphocytes. Recent studies have demonstrated that this defect is due to abolition of phosphorylation of key tyrosine residues within the linker region of PLCγ by Btk [23,24]. Btk is expressed in platelets and undergoes tyrosine phosphorylation upon activation of GPVI. Strikingly, XLA platelets exhibit reduced tyrosine phosphorylation of PLCγ2 and aggregation in response to collagen and CRP, whereas responses to other agonists are normal [25]. A defect in bleeding is not seen in these patients, most likely because of the partial nature of the impairment in response and redundancy between the pathways of platelet activation. The partial nature of the inhibition is due to co-expression of a second member of this family, Tec, as shown in studies on murine platelets deficient in the two kinases. Mice deficient in Btk exhibit a marked impairment in platelet activation downstream of CRP, whereas mice deficient in Tec exhibit a relatively mild defect [26]. A more pronounced defect is observed in mice deficient in both Btk and Tec [26]. It is not known whether the residual response that is seen in the absence of Btk and Tec is due to the presence of a third member of the Tec family in platelets or because a limited degree of activation of PLCγ2 can take place in the absence of this family of proteins.

The PI 3-kinase pathway is also required for maximal activation of PLCγ2 downstream of Syk in platelets, as shown using the structurally distinct inhibitors, wortmannin and Ly294002 [27]. This action is mediated through regulation of Tec family kinases and PLCγ2, both of which have PH domains that bind to the PI 3-kinase product, phosphatidylinositol 3,4,5-trisphosphate (PI 3,4,5P3), thereby facilitating their recruitment to the plasma membrane. The PI 3-kinase pathway regulates additional effector proteins in the cell and it is possible that one or more of these also plays a role in mediating activation of PLCγ, although this does not include the Akt family of serine-threonine kinases, which support aggregation by G protein-coupled receptors [28].

The class I PI 3-kinases convert phosphatidylinositol 4,5-bisphosphate into the second messenger PI 3,4,5P3, which supports the activation of Tec kinases and PLCγ2. Type I PI 3-kinases are further subdivided into IA and IB groups, which are regulated downstream of tyrosine kinases and G protein-coupled receptors, respectively. Class IA is made up of heterodimeric proteins, composed of regulatory and catalytic subunits. Five regulatory subunits (p85α, p55α, p50α, p85β, p85γ) derived from three gene products have been identified, with p85α, p55α and p50α being encoded by the same gene. Simultaneous deletion of p85α, p55α and p50α causes perinatal lethality in mice, whereas deletion of p85α alone does not. Studies on platelets from p85α−/− mice have demonstrated that this subunit appears to account for 95% of the PI 3-kinase activity in platelets and that it plays a key role in signaling downstream of CRP [29]. Platelets express all three catalytic isoforms of class IA PI 3-kinases, α, β and δ, although not all studies have been able to detect the presence of the α-isoform [29,30]. Studies on murine platelets deficient in PI 3-kinase-δ, or which express a catalytically inactive mutant of the kinase, have revealed a minor role for this isoform in response to stimulation by CRP, but not by G protein-coupled receptor agonists, suggesting that its role is masked by the presence of the other isoforms [30]. Similar studies on P110-α or PP110β-deficient platelets have yet to be performed, as deletion of either catalytic subunit causes embryonic lethality.

Platelets also express the two known isoforms of PLCγ, although PLCγ2 is expressed at a level approximately two orders of magnitude greater than PLCγ1 [31] and is the major isoform supporting activation downstream of GPVI, as shown using PLCγ2-deficient murine platelets [32]. In the absence of this isoform, mouse platelets undergo only a limited shape change response and very weak aggregation upon stimulation by CRP or convulxin [33]. This residual response is fully inhibited by the PLC inhibitor, U73122. The residual response in mouse platelets is believed to be mediated by PLCγ1, as this isoform undergoes tyrosine phosphorylation in response to GPVI receptor activation [33]. Confirmation of this will require studies on platelets from a mouse that has undergone a conditional deletion of PLCγ1, as deletion of this protein causes embryonic lethality. Interestingly, PLCγ1 does not undergo tyrosine phosphorylation in response to GPVI stimulation in human platelets [34] suggesting that it may not be functional in humans, in contrast to the situation in mice.

Thus, many of the proteins in the GPVI-signaling cascade have functional homologues, which have compensatory roles for each other. Two notable exceptions are Syk and SLP-76, which is consistent with the complete or almost complete ablation of response that is seen in the absence of either protein downstream of GPVI stimulation, respectively [35,36]. The observation that many proteins in the GPVI signaling cascade have functional counterparts has important implications for studies on mutant mouse models that have failed to detect a platelet phenotype, despite expression of the protein in wild-type platelets. However, alternative explanations for the apparent absence of function of a protein should also be considered. These include a role for the protein of interest in supporting activation by other platelet surface receptors; a role in megakaryocyte development/platelet formation; or a role in platelet activation in a more primitive organism. Indeed, it is noteworthy that no new classes of protein that play major roles in promoting activation of PLCγ isoforms downstream of ITAM receptors have been identified in recent years, suggesting that most of the important proteins have been identified. It remains an ongoing challenge to establish the precise role of each protein in supporting platelet activation, as this has important implications for development of antithrombotic agents and elucidation of the molecular basis of platelet function disorders.

We know much less about the mechanisms that inhibit activation of PLCγ2 by GPVI in platelets, although there appear to be at least three recognized pathways. Signalling by ITAM receptors is inhibited by engagement of receptors with one or more immunoreceptor tyrosine-based inhibition motifs (ITIMs) in their cytosolic tail. The tyrosine phosphatases SHP2 and SHP1 bind to phosphorylated ITIMs and inhibit activation of PLCγ, although the protein substrates that underlie this action remain unclear. Furthermore, this route of inhibition is also complicated by potential positive effects of the two phosphatases, as illustrated by the inhibition in response to CRP in mice platelets which are deficient or express a mutant form of SHP-1 [37].

PECAM-1 (or CD31) is the major ITIM-containing receptor on the platelet surface, being present at approximately 10 000 copies per cell (for reviews see Refs [38,39]). PECAM-1 strongly inhibits signaling by GPVI, although it can also inhibit activation by G protein-coupled receptors and by GPIb-IX-V [40–43]. PECAM-1 is activated by a homotypic interaction (i.e. PECAM-1 binds to itself), suggesting that its major physiologic role is to prevent activation of platelets on the endothelial surface, where it is expressed at approximately one million copies per cell.

Platelets have recently been shown to express two further ITIM-containing proteins. TREM-like transcript-1 (TLT-1) is present on the membrane of α-granules and becomes exposed on the platelet surface upon activation [44,45]. It appears however that TLT-1 contributes weakly to platelet activation, rather than mediating inhibition, with its overall functional significance appearing to be minor. The ITIM-containing receptor CD72 has a well-defined role in inhibiting signaling by the B-cell receptor and has recently been shown to be expressed on platelets along with its ligand, semaphorin 4D (CD100) [46]; its functional significance in platelets is not known.

The second route of inhibition of ITAM signaling cascades is through regulation of the Cbl family of adapter proteins, which also promote protein ubiquitination [47]. c-Cbl undergoes a marked increase in tyrosine phosphorylation upon engagement of GPVI and its absence leads to potentiation of the response to the Ig receptor [48], in line with the observations made for ITAM receptors in other hematopoietic cells. The molecular basis of the potentiation, and whether this is dependent on ubiquitination, is not known. The functional significance of the Cbl-inhibitory pathway is also unclear, although it is tempting to speculate that it represents a mechanism to reverse platelet activation under conditions of weak activation, perhaps for example, where there is only limited damage to the vessel wall.

The third mechanism of inhibition is through the action of non-SH2 domain-containing tyrosine phosphatases. In this case, however, it is unclear whether this is a result of the high level of constitutive activity or whether these have to be activated to mediate dephosphorylation. The adapter protein LAT has been shown to undergo transient phosphorylation following engagement of GPVI, and it has been proposed that this is mediated by protein tyrosine phosphatase 1B [49]. Platelets express several other tyrosine phosphatases, although very little is known about their roles in regulating signaling by GPVI and other platelet surface receptors, and this remains an important area for investigation.

Overall, the research over the last few years has further emphasized the similarity of the GPVI signaling cascade to that used by ITAM receptors in other cells. Furthermore, this work has also revealed that many of the proteins in the GPVI signaling cascade have functional homologs, while others do not appear to play a role in supporting activation, e.g. WASp [50]. These observations have important implications for the targetting of novel pharmaceuticals and understanding of disease processes involving ITAM-based signaling cascades in platelets and in other hematopoietic cells. The GPVI signaling pathways are summarized in Fig. 2.

Integrin signaling

  1. Top of page
  2. Abstract
  3. Introduction
  4. GPVI signaling
  5. Integrin signaling
  6. Signalling by other platelet integrins
  7. Thrombus formation in vitro and in vivo
  8. Conclusions
  9. Acknowledgements
  10. References

The early studies on tyrosine phosphorylation in platelets identified a key role for the tyrosine kinase Syk in signaling downstream of the major platelet integrin, αIIbβ3 [51]. This observation was subsequently supported by a series of elegant experiments in CHO cells from the group of Shattil which established critical roles for Syk, Vav and SLP-76 in mediating lamellipodia formation by the integrin [52,53]. However, it was not until studies were undertaken in mouse platelets deficient in SLP-76 and Syk that a functional role for this pathway during formation of these structures in platelets was established [21,54]. The study of Obergfell et al. [21] also used mutant mice and a Src kinase inhibitor to establish critical roles for Src and other members of the Src family in supporting recruitment and activation of Syk. The activation of Src kinases by αIIbβ3 has also recently been shown to lead to tyrosine phosphorylation of PLCγ2, an event that is vital for lamellipodia formation [55,56]. Thus, platelets express two distinct classes of surface receptor that signal through sequential activation of Src and Syk tyrosine kinases.

The Shattil group has recently elucidated several of the key events in the activation of Src and Syk kinases by αIIbβ3. Src is constitutively associated via its SH3 domain with the terminal portion of the β3-tail [57]. Clustering of αIIbβ3 leads to activation of Src through autophosphorylation at position 418 [57]. This leads to recruitment of Syk through an interaction mediated by its N-terminal SH2 domain and interdomain A region with the last 28 amino acids of the β3-tail [58,59]. Deletion of the two conserved tyrosines in the β3-tail prevents the interaction with Syk, as does tyrosine phosphorylation at these sites [58]. Consistent with this, the interaction between Syk and β3-tail is independent of the arginine residue in the N-terminal SH2 domain of Syk which is essential for binding to phosphotyrosine [58]. These observations emphasize the fact that the regulation of Src and Syk tyrosine kinases by integrin αIIbβ3 and GPVI signaling pathways are distinct, with the latter being dependent on ITAM phosphorylation.

It is predicted that Syk, SLP-76 and Vav play integral roles in the regulation of PLCγ2 by αIIbβ3, as is the case for GPVI, although this has not been proven. The two signaling cascades however can also be distinguished by their requirement for the tranmembrane adapter LAT, which does not undergo tyrosine phosphorylation downstream of αIIbβ3 [60,61]. LAT has two palmitoylated cysteine residues that target it to cholesterol-rich regions of the membrane, known as lipid rafts or gems. These regions are rich in receptors and signaling proteins, including the Src family kinases Fyn and Lyn, but not Src, which is one of the two members of this family to lack the palmitoylation sites required for targetting to rafts. GPVI has been reported to be constitutively expressed in membrane rafts or to be translocated into these regions upon activation [61–63]. The contrasting observations are almost certainly due to differences in the procedures used to isolate membrane rafts. Importantly, however, all of these studies demonstrated that signaling by GPVI is selectively inhibited by the cholesterol lowering reagent, β-methyl cyclodextrin, which disrupts these domains. In contrast, integrin αIIbβ3 is localized outside of membrane rafts [61], consistent with a role for Src in the proximal stages of its signaling cascade and the absence of involvement of LAT. These observations therefore reveal a remarkable degree of compartmentalization of signaling cascades by GPVI and αIIbβ3 within the platelet surface membrane.

In the absence of involvement of LAT, it is unclear as to how PLCγ2 is recruited to the membrane downstream of αIIbβ3. Furthermore, SLP-76 does not require the polyproline sequence that mediates binding to Gads to support αIIbβ3-mediated formation of lamellipodia in CHO cells [53]. The observation that tyrosine phosphorylation of PLCγ2 is markedly reduced, but not abolished, in the presence of an inhibitor of actin polymerization, indicates a role for the actin cytoskeleton in the possible recruitment of the phospholipase. This effect may however be a consequence of a decrease in the strength of the signal from the integrin, as a result of the impairment in spreading and therefore decrease in degree of ligand engagement [56].

The PI 3-kinase pathway also plays a critical role in signaling by integrin αIIbβ3 [64,65] and, in contrast to the results observed for GPVI, the p110δ catalytic subunit appears to play an important role in this process [30]. Interestingly, however, Btk and Tec do not appear to be required for spreading on fibrinogen, despite the fact that they undergo tyrosine phosphorylation downstream of the integrin [26]. This is surprising, given the critical role of Tec family kinases in regulating PLCγ2, although it remains possible that this has been missed because of the relatively weak signal that is required for formation of lamellipodia, bearing in mind that spreading was only monitored in this study at a single time point of 45 min. This could be addressed by monitoring adhesion and spreading using time-lapse, video microscopy [56,66].

A complete understanding of the role of the above signaling cascade in the activation of platelets by αIIbβ3 is complicated by stimulation of other signaling cascades by the integrin tail, as well as by the need for ‘inside-out’ signals to regulate ligand binding. The β3-tail contains two conserved tyrosines, both of which undergo phosphorylation in response to integrin activation [67]. The Src kinase Fyn has been proposed (as an unpublished observation) to mediate tyrosine phosphorylation of these two residues [68], although this has not yet been supported by a primary publication. The N-terminal tyrosine residue falls within a conserved NPXY motif that mediates binding to proteins with phosphotyrosine binding (PTB) domains, such as the adapter Dok2, which undergoes tyrosine phosphorylation downstream of the integrin [69–71]. The second tyrosine has a similar motif, NXXY, and has been shown to bind a subset of PTB domains, including Shc. The functional significance of phosphorylation on these two conserved tyrosines in these sequences is illustrated by the recurrent bleeding phenotype and impairment in clot retraction that is seen following their mutation to phenylalanine (the so-called diYF mutant mouse) [72]. The molecular basis of this defect has been attributed to the loss of binding of myosin to the phosphorylated β3 tail [73], in view of its role in supporting clot retraction, although it could also be due to loss of the interaction with Syk. It is not known whether Shc or Dok2 also contribute to this response, although inhibition of tyrosine phosphorylation of Shc in the diYF mutant mouse has been reported [69].

The β3-integrin tail binds to several additional signaling proteins, many of which are also implicated in mediating outside-in signaling by the integrin. The β3-tail supports the formation of a complex between the adapter Rack1 and protein kinase Cβ, consistent with a role for the latter in mediating platelet spreading [56,74]. Bioluminescence resonance energy transfer (BRET) has been used to demonstrate the presence of distinct signaling complexes of integrin αIIbβ3 and Src with either focal adhesion kinase (FAK) or Syk in CHO cells [75]. Strikingly these complexes are distributed within distinct regions of the activated cell, suggesting that they have different functions. The identification of these two complexes is also consistent with previous observations in CHO cells, in which tyrosine phosphorylation of FAK was observed following disruption of binding of Syk to β3-tail [58]. The role of tyrosine phosphorylation of the β3-tail in these two pathways is unclear.

Thus, αIIbβ3 activates both Syk-dependent and independent signaling cascades, several of which are illustrated in Fig. 3. Significantly, many of these cascades cannot be regulated simultaneously as, for example, tyrosine phosphorylation of β3-tail, prevents the interaction with Syk.


Figure 3. Integrin αIIbβ3 regulates Syk-dependent and -independent cascades. Outside-in signaling through ligand engagement or clustering of integrin αIIbβ3 generates Syk-dependent and -independent intracellular signaling cascades. (A) Src-dependent activation of Syk leads to activation of PLCγ2 through a pathway that is likely to be dependent on SLP-76 and Vav. (B) Tyrosine phosphorylation of two conserved tyrosines, possibly by Fyn, leads to binding and tyrosine phosphorylation of the adapters Shc and Dok2, and also recruitment of myosin. (C) αIIbβ3 and Src have been shown to form an intracellular complex with focal adhesion kinase that is independent of Syk. It is not known whether the interaction between the three proteins is direct. (D) Amino acid sequence of β3-tail.

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The last few years have therefore seen significant advances in our understanding of integrin αIIbβ3 signaling, although the challenge remains to establish the stoichiometry of the interactions; to determine the way that these signals interact with each other; and their involvement in functional responses, such as spreading and clot retraction, and the roles that these responses themselves play in supporting thrombus formation. These are far from straightforward challenges however because of the complex nature of integrin function and the difficulties in studying outside-in signaling in isolation from inside-out signals. Specific issues include (i) multiplicity of the signals from the integrin; (ii) the need for inside-out signals to activate the integrin, (iii) the cooperative nature of integrin-based signals with those of other receptors [76], (iv) the degree to which data from cell line models can be extrapolated to the platelet and (v) separating the critical role of the integrin in supporting aggregation from other responses. For example, using sophisticated, real-time calcium imaging technology, a role for integrin αIIbβ3 has been proposed in promoting thrombus growth in combination with activation of P2Y12 ADP receptors through a process known as intercellular calcium communication (ICC) [77]. It is difficult, however, to establish the full significance of this mechanism because of the critical role of the integrin in platelet aggregation. Similarly, it is unclear whether the role of the integrin in supporting clot retraction is primarily mechanical, in that it provides a link with the cytoskeleton and the fibrin rich matrix, or if the integrin also generates intracellular signals that support this response. It is however clear that integrin-mediated spreading plays a critical role in supporting platelet adhesion and platelet aggregation at arterial rates of flow on a collagen matrix as these responses are markedly impaired in mice deficient in the small G protein Rac, which is essential for lamellipodia formation (O.J.T. McCarty, J.M. Auger and S. P. Watson, unpublished data).

Signalling by other platelet integrins

  1. Top of page
  2. Abstract
  3. Introduction
  4. GPVI signaling
  5. Integrin signaling
  6. Signalling by other platelet integrins
  7. Thrombus formation in vitro and in vivo
  8. Conclusions
  9. Acknowledgements
  10. References

It is important to consider the extent to which the above signaling events can be extrapolated to the other platelet integrins, bearing in mind that they are expressed at between 0.5% and 3% of the level of αIIbβ3. The most thoroughly studied of the other platelet integrins is the collagen receptor α2β1. Recent evidence suggests that integrin α2β1 also signals through sequential activation of Src and Syk family tyrosine kinases leading to activation of PLCγ2 and formation of lamellipodia [78,79]. A complete understanding of the significance of signaling by integrin α2β1 in mediating responses to collagen is unclear, however, because of the much greater signaling strength of GPVI and the existence of other signaling receptors for collagen of uncertain significance [80]. It is nevertheless clear that integrin α2β1 plays an important role in supporting adhesion and thrombus stability on collagen under flow conditions [81–84], and it will be important to establish the contribution that signaling plays in these events.

In contrast to the ability of α2β1 to mediate full platelet spreading, it has recently been reported that engagement of integrin α5β1 by fibronectin (in the presence of an αIIbβ3-receptor antagonist) generates formation of filopodia but not lamellipodia [66]. This observation is particularly interesting in that integrin α5β1 is expressed at a similar level to α2β1, suggesting either a difference in the two β1-signaling cascades or the involvement of an additional receptor in the response to collagen. In this context, it is interesting that engagement of αvβ3 by vitronectin, which is expressed at a similar level to the two β1-integrins, induces formation of filopodia but not lamellipodia in platelets, in contrast to αIIbβ3 engagement [66].

It is now recognized that many of the features in the signaling pathway used by αIIbβ3 are shared with other platelet integrins, but the overall significance of signaling by these integrins remains unclear because of the relatively weak nature of the signal, which is a consequence of their relatively low level of expression.

Thrombus formation in vitro and in vivo

  1. Top of page
  2. Abstract
  3. Introduction
  4. GPVI signaling
  5. Integrin signaling
  6. Signalling by other platelet integrins
  7. Thrombus formation in vitro and in vivo
  8. Conclusions
  9. Acknowledgements
  10. References

It is essential to establish the significance of the above biochemical and functional observations in more relevant physiologic models, most notably with respect to the influence of the range of rates of shear found in physiologic and pathologic conditions. This can be achieved using in vitro flow models, which allow the use of a defined extracellular matrix and controlled rates of flow, and in animal models in vivo.

A frequently used experimental model is that of flowing whole blood over a monolayer of collagen at high shear, as this shares many of the features that are seen in vivo [38]. The initial event is tethering of platelets via the binding of VWF, which is immobilized on the collagen surface, to platelet GPIb-IX-V. This interaction is insufficient to promote stable adhesion over short periods of time due to the fast off-rate of binding of VWF to GPIb-IX-V [85]. Instead, the generation of powerful intracellular signals by GPVI stimulates integrin activation and thereby leads to stable adhesion, through binding of collagen to integrin α2β1 and VWF to αIIbβ3 [86–88]. In comparison, stable adhesion is only seen on an immobilized VWF surface (in the absence of collagen) after several minutes. This observation is consistent with the one that GPIb-IX-V generates a relatively weak intracellular signal, although it is possible that this has a greater physiologic significance in cooperation with other receptors [38,77]. It is also now recognized that limited stable adhesion can occur directly to integrin α2β1 in the absence of intracellular signals [84,89], a process that may be mediated through the α2β1-specific GFOGER sequence in collagen [82]. Adherent platelets generate filopodia and lamellipodia, which serves to strengthen their contact with the extracellular matrix and thereby enable them to withstand the increasing shear forces which are generated as the thrombus develops.

As indicated above, the platelet monolayer serves as a base for platelet aggregation, a process that is dependent on binding of bivalent fibrinogen to integrin αIIbβ3. Aggregation is mediated by a process involving capture of platelets by VWF bound to αIIbβ3 and activation of αIIbβ3 by the secondary mediators, ADP and thromboxane A2, and by the product of the coagulation cascade, thrombin [83,88,90]. Thrombus stability is further enhanced by clot retraction, which is dependent on fibrin engagement of integrin αIIbβ3 and stimulation of contraction [91].

The role of Src kinases and PLCγ2 in supporting platelet adhesion and platelet aggregate formation on a VWF/collagen surface at high shear is illustrated by the complete abolition of aggregate formation and dramatic reduction in adhesion observed through use of a Src kinase inhibitor [84] and in mouse platelets deficient in the phospholipase [33], respectively. The slightly greater level of adhesion that is seen in the absence of PLCγ2 than in platelets deficient in the GPVI/FcR γ-chain complex suggests that a residual degree of signaling occurs in response to collagen, which is most likely mediated by PLCγ1 [33]. The reduction in adhesion and aggregate formation is due to loss of signals from GPVI, rather than from integrin αIIbβ3 or α2β1, as shown by studies on FcR γ-chain-deficient mice platelets that express an FcR γ-chain transgene in which the ITAM tyrosines have been replaced by phenyalanines, thereby inhibiting signaling but not GPVI expression [92]. A reduction in adhesion and aggregation on VWF/collagen is also seen in Vav1/Vav3-deficient platelets, consistent with its role in the activation of PLCγ2 [22].

It remains unclear as to how far results from this model can be used to predict events that underlie thrombus formation in vivo, bearing in mind that platelets are exposed to additional proteins following damage to the subendothelial matrix. Several models of thrombus formation have therefore been developed in mice, each with their own strengths and limitations [93]. A recently introduced model is that of monitoring thrombus formation in real time using fluorescent microscopy following lesion of the endothelial layer with a nitrogen dye laser [94]. Using this approach, we have demonstrated a dramatic reduction in thrombus formation in mice deficient in PLCγ2, together with an increased rate of embolization of the newly formed thrombus (N. Kalia and B. Atkinson, unpublished data). Further work is required to establish the roles of GPVI, αIIbβ3 and other platelet receptors in supporting this response. It will also be important to extend this approach to additional signaling proteins, including Vav and Tec family proteins.


  1. Top of page
  2. Abstract
  3. Introduction
  4. GPVI signaling
  5. Integrin signaling
  6. Signalling by other platelet integrins
  7. Thrombus formation in vitro and in vivo
  8. Conclusions
  9. Acknowledgements
  10. References

We now have a firm understanding of the key features of the GPVI signaling cascade in platelets and have identified many of the similarities and differences with the cascade used by integrin αIIbβ3. PLCγ2 is recognized as a central target for these two signaling cascades, and also those used by other platelet glycoprotein receptors, including GPIb-IX-V [95]. There are, however, significant differences in the degree of activation of PLCγ2, with GPVI being considerably more powerful than the other receptors. This may be explained by fundamental differences in signaling pathways, such as participation of key regulatory molecules e.g. LAT, and sites of phosphorylation of PLCγ2 [96].

These recent observations have established a key role for PLCγ2 in supporting thrombus formation in vivo. It will be important to extend this to other signaling molecules in our attempts to understand the events that underlie thrombus formation in healthy and diseased vessels. The potential for signaling molecules such as PLCγ2 to serve as targets for development of new types of antithrombotics is worthy of consideration.


  1. Top of page
  2. Abstract
  3. Introduction
  4. GPVI signaling
  5. Integrin signaling
  6. Signalling by other platelet integrins
  7. Thrombus formation in vitro and in vivo
  8. Conclusions
  9. Acknowledgements
  10. References

SPW holds a British Heart Foundation Chair. We thank Drs Hughan, Kalia and Tomlinson for their constructive comments on this text. We also thank present and past members of the laboratory whose work has contributed significantly to the ideas that are discussed in this review. The authors’ research is supported by the British Heart Foundation, Wellcome Trust and Medical Research Council.


  1. Top of page
  2. Abstract
  3. Introduction
  4. GPVI signaling
  5. Integrin signaling
  6. Signalling by other platelet integrins
  7. Thrombus formation in vitro and in vivo
  8. Conclusions
  9. Acknowledgements
  10. References
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