Platelets perform a central role in haemostasis and thrombosis. They adhere to subendothelial collagens exposed at sites of blood vessel injury via the glycoprotein (GP) Ib-V-IX receptor complex, GPVI and integrin α2β1. These receptors perform distinct functions in the regulation of cell signalling involving non-receptor tyrosine kinases (e.g. Src, Fyn, Lyn, Syk and Btk), adaptor proteins, phospholipase C and lipid kinases such as phosphoinositide 3-kinase. They are also coupled to an increase in cytosolic calcium levels and protein kinase C activation, leading to the secretion of paracrine/autocrine platelet factors and an increase in integrin receptor affinities. Through the binding of plasma fibrinogen and von Willebrand Factor to integrin αIIbβ3, a platelet thrombus is formed. Although increasing evidence indicates that each of the adhesion receptors GPIb-V-IX and GPVI and integrins α2β1 and αIIbβ3 contribute to the signalling that regulates this process, the individual roles of each are only beginning to be dissected. By contrast, adhesion receptor signalling through platelet endothelial cell adhesion molecule 1 (PECAM-1) is implicated in the inhibition of platelet function and thrombus formation in the healthy circulation. Recent studies indicate that understanding of platelet adhesion signalling mechanisms might enable the development of new strategies to treat and prevent thrombosis.

Introduction

Platelets are small, anucleate blood cells derived from megakaryocytes. They provide a first line of defence following injury, forming thrombi that patch-up damaged tissue and thereby playing an indispensable role in haemostasis. However, inappropriate platelet activation can lead to thrombosis, myocardial infarction and strokes. Platelets are also believed to be involved in the development of atherosclerosis in coronary or carotid arteries, which is commonly the trigger for thrombosis.

Platelets possess several cell-surface receptors that allow them to adhere to sites of tissue damage and spread to form a monolayer of cells that covers the exposed tissue. Spreading is accompanied by the secretion or synthesis of several prothrombotic factors, such as ADP, serotonin and thromboxane A2, which act in an autocrine/paracrine fashion and activate or prime approaching platelets (Ruggeri, 2002). During platelet activation, inside-out signalling upregulates the affinity of several platelet integrins, including integrin αIIbβ3 (Calderwood, 2004; Liddington and Ginsberg, 2002; Shattil et al., 1998). This binds to the bivalent ligand fibrinogen, which is present in the plasma and is released by activated platelets. The resulting platelet aggregation leads to the assembly of a platelet thrombus.

Although platelets do not directly activate the coagulation pathways, they are vital for effective blood coagulation, providing a surface for the assembly of the prothrombinase complex. This `procoagulant' property depends on exposure of aminophospholipids such as phosphatidylserine on the cell surface, and the release of phospholipid microparticles (Heemskerk et al., 2002). Coagulation is precipitated by the cleavage of fibrinogen by the serine protease thrombin, an end-product of the clotting pathways, and subsequent formation of an insoluble polymeric fibrin mesh. Since thrombin is generated on the surface of activated platelets, fibrin is deposited in the platelet thrombus as it is assembled (Falati et al., 2002). Thrombin is also a powerful platelet agonist that can stimulate platelet aggregation and thrombus formation.

Here, I discuss our current understanding of the initial signalling responses of platelets following tissue injury, focusing on signalling stimulated by non-integrin adhesion receptors and mechanisms for the negative regulation of platelet function by platelet endothelial cell adhesion molecule 1 (PECAM-1). Reviews that cover other aspects of platelet signalling may be found elsewhere (Jackson et al., 2003; Nieswandt and Watson, 2003).

Platelet adhesion receptors

When the integrity of the vascular system is breached, platelets are exposed to components of the extracellular matrix (ECM) present in the blood vessel wall and beyond. Platelets can interact directly or indirectly with several ECM proteins, but of principal importance are the collagens (Farndale et al., 2004). Humans possess at least 25 forms of collagen (Hashimoto et al., 2002), and several of these (I, III, IV, V, VI, VIII, XII, XIII and XIV) are present in the blood vessel wall (Barnes and Farndale, 1999). In addition, type IV collagen is present in the subendothelial basement membrane.

The GPIb-V-IX complex

The initial entrapment of platelets on subendothelial collagens requires the plasma protein von Willebrand factor (VWF), which under the shear stress conditions present in arteries and small arterioles binds simultaneously to collagen and the platelet glycoprotein (GP) complex GPIb-V-IX, or to the integrin αIIbβ3 in its activated conformation (Fig. 1) (Alevriadou et al., 1993; Ruggeri, 1997; Savage et al., 1996; Sixma et al., 1997). VWF-dependent interactions have a fast off rate and cannot support the assembly of a platelet thrombus. These interactions are superseded by more-stable binding of collagen to platelet collagen receptor, principally integrin α2β1 and GPVI (Moroi et al., 1996; Saelman et al., 1994b; Staatz et al., 1989) (reviewed by Farndale et al., 2004; Jackson et al., 2003; Nieswandt and Watson, 2003; Ruggeri, 2002).

Fig. 1.

Stages in the development of a platelet thrombus on collagen exposed at sites of injury. The initial interaction of platelets with subendothelial collagens under high shear conditions present in the arterial circulation is indirectly mediated by von Willebrand factor, which binds collagen and platelet GPIb. This unstable interaction facilitates transient tethering and rolling. GPIb-mediated adhesion is superseded by more-stable binding to collagen by GPVI and integrin α2β1. This, together with GPIb, stimulates platelet signalling that results in shape change and spreading, and the secretion and release of multiple prothrombotic factors. Integrin affinity becomes upregulated through inside-out signalling, resulting in fibrinogen-mediated platelet aggregation through binding to integrin αIIbβ3, and adhesion is stabilized by enhanced binding of collagen and von Willebrand factor to integrins α2β1 and αIIbβ3, respectively.

Fig. 1.

Stages in the development of a platelet thrombus on collagen exposed at sites of injury. The initial interaction of platelets with subendothelial collagens under high shear conditions present in the arterial circulation is indirectly mediated by von Willebrand factor, which binds collagen and platelet GPIb. This unstable interaction facilitates transient tethering and rolling. GPIb-mediated adhesion is superseded by more-stable binding to collagen by GPVI and integrin α2β1. This, together with GPIb, stimulates platelet signalling that results in shape change and spreading, and the secretion and release of multiple prothrombotic factors. Integrin affinity becomes upregulated through inside-out signalling, resulting in fibrinogen-mediated platelet aggregation through binding to integrin αIIbβ3, and adhesion is stabilized by enhanced binding of collagen and von Willebrand factor to integrins α2β1 and αIIbβ3, respectively.

Binding of VWF to GPIb-V-IX upregulates integrin αIIbβ3 affinity (Asazuma et al., 1997; Kasirer-Friede et al., 2004; Milner et al., 1998; Munday et al., 2000; Nesbitt et al., 2002; Torti et al., 1999; Yuan et al., 1999). The integrin can then bind to VWF, thereby enhancing adhesion, and contributing to thrombus formation by binding to fibrinogen.

Integrin α2β1

Integrin α2β1 was the first platelet collagen receptor to be identified and binds to collagen in a Mg2+-dependent manner (Kunicki et al., 1988; Nieuwenhuis et al., 1985; Santoro, 1986; Santoro et al., 1988; Sixma et al., 1995; Sixma et al., 1997). Integrin α2β1 does not stimulate tyrosine kinase activity, which is required for collagen-induced platelet activation. Santoro et al. therefore proposed a two-site–two-step model of platelet activation (Santoro et al., 1991) in which integrin α2β1 stabilizes interactions with collagen, allowing it to interact with a second collagen receptor that can activate tyrosine-kinase-dependent signalling. This model, in which platelet adhesion and activation are considered distinct events, is supported by more-recent studies (Jung and Moroi, 1998; Jung and Moroi, 2000; Moroi et al., 2000; Siljander et al., 2004).

In common with other integrins, the affinity of α2β1 for collagen is increased by inside-out signalling (Inoue et al., 2003; Jung and Moroi, 1998; Jung and Moroi, 2000; Nieswandt et al., 2001a). Thereby, platelet activation leads to increased affinity and increased adhesion stability.

Glycoprotein VI (GPVI)

The second collagen receptor is a multi-protein complex containing GPVI and the Fc receptor γ-chain (FcRγ)* (Gibbins et al., 1997; Tsuji et al., 1997). GPVI was originally identified as a potential collagen receptor in patients expressing low levels of the protein, who display mild bleeding diatheses (Arai et al., 1995; Moroi et al., 1989; Ryo et al., 1992), and was subsequently cloned and characterized (Clemetson et al., 1999; Ezumi et al., 2000; Jandrot-Perrus et al., 2000). It associates noncovalently with FcRγ (Gibbins et al., 1997; Tsuji et al., 1997), and anti-GPVI F(ab')2 fragments stimulate platelet activation (Gibbins et al., 1997; Sugiyama et al., 1987). Studies of knockout or knockdown mice support the idea that this receptor is the principal activatory collagen receptor (Kato et al., 2003; Massberg et al., 2003; Nieswandt et al., 2001a; Schulte et al., 2003). Mutagenesis studies based on sequence differences between human and mouse GPVI, and the generation of an inhibitory phage antibody against GPVI, have enabled the collagen-binding surface in GPVI to be defined (Smethurst et al., 2004).

*

The FcRγ is a component of the multi-subunit high-affinity receptor for immunoglobulin (Ig)E, FcϵRI (Blank et al., 1989; Kuster et al., 1990), and the IgG receptors FcγRI and FcγRIII (van de Winkel and Capel, 1993). The FcRγ is noncovalently associated with these receptors and contains an immunoreceptor tyrosine-based activation motif (ITAM) (Reth, 1989), which becomes phosphorylated on receptor ligation and clustering. The IgG receptor FcγRIIA does not couple to FcRγ but contains a similar ITAM in its cytoplasmic tail (van de Winkel and Capel, 1993).

The complementary roles of α2β1 and GPVI

Much recent discussion has focused on the relative contributions of α2β1 and GPVI in platelet adhesion to collagen and to thrombus formation. Mouse platelets that lack GPVI as a consequence of intravenous injection of an anti-mouse GPVI monoclonal antibody or FcRγ gene ablation [GPVI expression is dependent on FcRγ expression (Nieswandt et al., 2000)] are resistant to activation by collagen and collagen-related peptide (which is a GPVI selective ligand, see below) (Nieswandt et al., 2001b) or adhesion to collagen under static and flow conditions (Nieswandt et al., 2001a). These mice are resistant to lethal pulmonary thromboembolism induced by collagen and adrenaline infusion, and tail bleeding times are moderately extended (Nieswandt et al., 2001b). Integrin-β1-null platelets have been reported to aggregate in response to collagen, although this is slightly delayed (Nieswandt et al., 2001a). At low (150 s–1) and high (1000 s–1) levels of shear stress, they adhere to soluble collagen normally. The authors of these studies have therefore proposed that GPVI, and not integrin α2β1, could thus be essential for the platelet adhesion to collagen, and that the initial collagen interaction is predominantly through GPVI, which results in cell signalling that upregulates α2β1 affinity (Nieswandt et al., 2001a). However, in contrast to β1-deficient-platelets, α2-deficient platelets fail to adhere to fibrillar collagen under low shear stress (Chen et al., 2002) and abnormal interaction of α2-deficient platelets with soluble collagen has been reported (Holtkotter et al., 2002).

Transgenic mice in which GPVI is deleted have been generated recently, and platelets from these mice fail to respond to collagen in aggregation assays (Kato et al., 2003). However, tail bleeding times in these mice are essentially normal. Moreover, in perfusion experiments at high shear stress, platelets lacking GPVI adhere to insoluble type I collagen but fail to form thrombi. These results closely resemble those obtained for human GPVI-deficient platelets in similar assays (Moroi et al., 1996). Similar observations have also been reported for normal platelets and a function-blocking anti-GPVI antibody (Siljander et al., 2004), suggesting that GPVI and α2β1 might play complementary roles in which α2β1 is able to bind to collagen before platelet activation and GPVI is required for thrombus formation (Kuijpers et al., 2003).

The reasons for discrepancies between some of these studies remain to be resolved, although these are likely to be due to different experimental systems used. However, the studies do point towards a pivotal role of GPVI in haemostasis and thrombosis. Since inhibition and in vivo depletion of GPVI are well tolerated in mice, targeting GPVI might provide new avenues for anti-thrombotic therapies.

More platelet collagen receptors?

A third functional collagen receptor on platelets might also exist. For example, in platelets lacking the GPVI-FcRγ complex, collagen stimulates low levels of protein tyrosine phosphorylation. Furthermore, in the presence of α2β1- and GPV-blocking antibodies, mouse platelets that possess approximately 20% of the normal levels of GPVI-FcRγ respond to collagen but not to collagen-related peptide (a GPVI-selective ligand, see below) (Poole et al., 1997; Snell et al., 2002). Several additional putative platelet collagen receptors have been reported, including 68 kDa (Monnet et al., 2001; Monnet and Fauvel-Lefeve, 2000) and 65 kDa (Chiang et al., 1997) binding proteins for type III collagen, and a 47 kDa type I collagen-binding species (Chiang et al., 2002). It is unclear whether any of these contribute to platelet signalling, although some may have supporting roles and modulate GPVI-mediated responses. CD36 was proposed to be a platelet collagen receptor, but since CD36-deficient platelets exhibit normal responses to collagen, this is unlikely (Daniel et al., 1994; Saelman et al., 1994a; Yamamoto et al., 1992). The most compelling evidence for a third collagen receptor is from studies of platelets from GPV-null mice, which display decreased adhesion and aggregation responses to collagen (Kahn et al., 1999; Ramakrishnan et al., 1999). Such platelets curiously demonstrate enhanced responses to thrombin (Ramakrishnan et al., 1999) and therefore determination of the relevance in vivo of GPV in collagen responses has not been possible.

Cell signalling mechanisms

Since platelets are anucleate, most in vitro molecular biology techniques are not applicable to these cells. Although the production of platelet-like particles from megakaryocytes in culture is possible, this is not routinely achieved and the low yield prevents extensive functional and biochemical analysis (Hartwig and Italiano, 2003; Italiano et al., 1999). Much research performed on primary platelets has used biochemical techniques, selective inhibitors of signalling enzymes, examination of naturally occurring mutations, and comparison with signalling mechanisms in other cells. In recent years, transgenic mouse models have become increasingly important.

Signalling through GPIb

As described below, the initial interaction of the GPIb-V-IX complex with VWF stimulates platelet signalling, which leads to the secretion of granules and the upregulation of integrin affinity. VWF binding requires shear stress, but shear-induced conditions for GPIb-VWF binding appear to be mimicked by the conformational modulator botrocetin and the antibiotic ristocetin, molecules that have been used in many GPIb-V-IX signalling studies. However, several studies have examined GPIb-V-IX in vitro using flow-based assay systems (Nesbitt et al., 2002; Yap et al., 2000). GPIb binding is associated with the stimulation of tyrosine kinase signalling (Asazuma et al., 1997; Ozaki et al., 1995; Razdan et al., 1994). Principal players in this pathway include the non-receptor tyrosine kinases Src, Fyn, Lyn and Syk, phospholipase Cγ2 (PLCγ2), and adaptor proteins such as Shc, linker for activation of T cells (LAT) and SLP-76 (Asazuma et al., 1997; Falati et al., 1999; Jackson et al., 1994; Marshall et al., 2002; Torti et al., 1999; Wu et al., 2001). Exactly how these components cooperate in GPIb-V-IX signalling is not known, but there is good evidence that it might be similar to GPVI signalling in platelets (described in detail below; Fig. 2). GPIb might also activate platelets by triggering Src-family-kinase-mediated phosphorylation of FcRγ and FcγRIIA, receptors with which GPIb physically associates (Canobbio et al., 2001; Falati et al., 1999; Sullam et al., 1998; Torti et al., 1999; Wu et al., 2001).

Fig. 2.

Model of platelet signalling stimulated by collagen and von Willebrand factor (VWF). Collagen binding results in GPVI clustering and tyrosine phosphorylation of the Fc receptor γ-chain (FcRγ) by the Src-family kinases Lyn and Fyn. This results in the binding of the tyrosine kinase Syk, which becomes tyrosine phosphorylated and activated. This leads to the tyrosine phosphorylation of the transmembrane adaptor protein LAT, which functions to assemble a complex of signalling proteins. Phosphoinositide 3-kinase (PI3K) is recruited to LAT and, through the generation of, among other products, phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3, PIP3 in figure], influences the recruitment and activation of phospholipase Cγ2 (PLCγ2). PLCγ2 cleaves phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2, PIP2 in figure] to generate inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] and diacylglycerol (DAG). Ins(1,4,5)P3 and DAG are responsible for the mobilization of calcium from intracellular stores and the activation of isoforms of protein kinase C (PKC), respectively, both of which lead to secretion and aggregation. PI3K activity also results in the regulation of protein kinase B (PKB), phosphoinositide-dependent kinase 1 (PDK1) and integrin-linked kinase (ILK), which are believed to be involved in integrin regulation. PKB also inhibits glycogen synthase kinase 3 (GSK3), which might contribute to a negative-feedback pathway mediated by PKB. The adaptor proteins Gads, SLP-76 and SLAP-130, and the Rho GTP exchange factor Vav, are also recruited to LAT, although the significance of this is unclear. The binding of VWF to GPIb-V-IX results in calcium fluxes that lead to the upregulation of integrin αIIbβ3 affinity, although the signalling pathway underlying this has not yet been defined. A proposed model based upon current data implicates several molecules involved in GPVI signalling in this process. These include FcRγ, Fyn, Lyn, Syk, LAT, PLCγ2, SLP-76, PI3K and PKC. It is possible that a signalling complex similar to that formed in GPVI signalling might mediate GPIb-V-IX signalling as shown, although this model is not confirmed. The roles of other GPIb-V-IX-binding proteins such as filamin, 14-3-3ζ and calmodulin (CAM) have yet to be determined.

Fig. 2.

Model of platelet signalling stimulated by collagen and von Willebrand factor (VWF). Collagen binding results in GPVI clustering and tyrosine phosphorylation of the Fc receptor γ-chain (FcRγ) by the Src-family kinases Lyn and Fyn. This results in the binding of the tyrosine kinase Syk, which becomes tyrosine phosphorylated and activated. This leads to the tyrosine phosphorylation of the transmembrane adaptor protein LAT, which functions to assemble a complex of signalling proteins. Phosphoinositide 3-kinase (PI3K) is recruited to LAT and, through the generation of, among other products, phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3, PIP3 in figure], influences the recruitment and activation of phospholipase Cγ2 (PLCγ2). PLCγ2 cleaves phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2, PIP2 in figure] to generate inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] and diacylglycerol (DAG). Ins(1,4,5)P3 and DAG are responsible for the mobilization of calcium from intracellular stores and the activation of isoforms of protein kinase C (PKC), respectively, both of which lead to secretion and aggregation. PI3K activity also results in the regulation of protein kinase B (PKB), phosphoinositide-dependent kinase 1 (PDK1) and integrin-linked kinase (ILK), which are believed to be involved in integrin regulation. PKB also inhibits glycogen synthase kinase 3 (GSK3), which might contribute to a negative-feedback pathway mediated by PKB. The adaptor proteins Gads, SLP-76 and SLAP-130, and the Rho GTP exchange factor Vav, are also recruited to LAT, although the significance of this is unclear. The binding of VWF to GPIb-V-IX results in calcium fluxes that lead to the upregulation of integrin αIIbβ3 affinity, although the signalling pathway underlying this has not yet been defined. A proposed model based upon current data implicates several molecules involved in GPVI signalling in this process. These include FcRγ, Fyn, Lyn, Syk, LAT, PLCγ2, SLP-76, PI3K and PKC. It is possible that a signalling complex similar to that formed in GPVI signalling might mediate GPIb-V-IX signalling as shown, although this model is not confirmed. The roles of other GPIb-V-IX-binding proteins such as filamin, 14-3-3ζ and calmodulin (CAM) have yet to be determined.

The binding of VWF to GPIb under shear stress can stimulate calcium mobilization (Kroll et al., 1991; Mazzucato et al., 2002; Milner et al., 1998; Nesbitt et al., 2002; Yap et al., 2000), protein kinase C (PKC), protein kinase G (PKG) (Li et al., 2003; Yap et al., 2000), phosphoinositide 3-kinase (PI3K) (Jackson et al., 1994; Munday et al., 2000; Yap et al., 2000) and cytoskeletal rearrangements (Torti et al., 1999; Yuan et al., 1999). VWF binding also upregulates integrin αIIbβ3 affinity indirectly through the stimulation of ADP secretion (Moake et al., 1988). Other studies have indicated that GPIb-V-IX controls upregulation of integrin αIIbβ3 affinity through FcRγ-independent interactions with signalling molecules and sequentially activates Src-family kinases, calcium oscillations, PI3K and PKC. Direct interactions of GPIb-V-IX with molecules such as calmodulin and 14-3-3ζ, and with the platelet cytoskeleton, have also been implicated in this (Andrews et al., 1998; Andrews et al., 2001; Cunningham et al., 1996; Kasirer-Friede et al., 2004; Munday et al., 2000).

For some time, controversy has surrounded the question of whether the GPIb-V-IX complex signals at all, and some studies have failed to show signalling by this receptor complex (Kuwahara et al., 1999). This is likely to reflect differences between studies with respect to the array of preparations and species of VWF, GPIb-V-IX-binding venom proteins and peptides, conformational modulators, experimental strategies and cell types used. Given the increasing literature in this area, it is likely that GPIb-V-IX does signal, although in comparison with what is known about GPVI-mediated signalling (see below), our understanding is much less developed. Although many molecules are implicated in GPIb-V-IX signalling, these have yet to be assembled into a defined signalling pathway.

Signalling through GPVI

A single collagen fibre can bind simultaneously to multiple different receptors and collagen-binding proteins on the platelet surface, which complicates the analysis of signalling by individual receptors. The analysis of differential adhesion and aggregatory properties of cyanogen bromide fragments of collagen led to the realization that different collagen receptors bind to distinct sequences within collagen fibres (Morton et al., 1989; Morton et al., 1994). A combination of collagen peptide functional analysis and the development of methods for the synthesis of triple helical peptides in which to present receptor-binding sequences have enabled the development of GPVI- and α2β1-selective ligands. Collagen-related peptides (CRPs) containing a repeated GPO sequence (single-letter amino acid code; O represents hydroxyproline) bind specifically to GPVI and are highly potent platelet agonists, and a GFOGER peptide supports α2β1-mediated adhesion (Asselin et al., 1997; Kehrel et al., 1998; Morton et al., 1995; Knight et al., 1998). These reagents, together with activatory and inhibitory antibodies to GPVI (Nieswandt et al., 2000; Smethurst et al., 2004; Sugiyama et al., 1987) and α2β1 (Polanowska-Grabowska and Gear, 1992; Stevens et al., 2004), and snake venom proteins, particularly convulxin (Batuwangala et al., 2004; Francischetti et al., 1997), have proven invaluable tools to study collagen-receptor-mediated signalling and function. However, it should be noted that it has recently been reported that, in addition to binding GPVI, convulxin also binds GPIb (Kanaji et al., 2003).

Exposure of platelets to collagen surfaces is believed to result in clustering of GPVI, which triggers the tyrosine phosphorylation of FcRγ (Gibbins et al., 1996; Gibbins et al., 1997; Poole et al., 1997; Tsuji et al., 1997) (Fig. 2). Several reports indicate that GPVI signalling is influenced strongly by glycolipid-enriched microdomains (GEMS, rafts) in the plasma membrane, although there is disagreement between authors on whether GPVI is constitutively associated with, or recruited to, lipid rafts (Ezumi et al., 2002; Locke et al., 2002; Wonerow et al., 2002).

The Src-family tyrosine kinases Fyn and Lyn, which are physically associated with GPVI, are responsible for FcRγ phosphoryation (Briddon and Watson, 1999; Ezumi et al., 1998; Quek et al., 2000; Suzuki-Inoue et al., 2002). They target conserved tyrosine residues within the immunoreceptor tyrosine-based activation motif [ITAM; consensus: Y-X-X-L/I-X6-8-Y-X-X-L/I, where X denotes any amino acid (Reth, 1989)] in the cytoplasmic tail of FcRγ (Gibbins et al., 1996; Poole et al., 1997). The phosphorylated ITAM provides a docking site for Syk, which binds specifically through tandem Src-homology 2 (SH2) domains (Benhamou et al., 1993; Shiue et al., 1995). As a consequence, Syk becomes tyrosine phosphorylated, probably by autophosphorylation, and activated. A substrate for Syk is the adaptor LAT (Zhang et al., 1998), which possesses multiple phosphorylation sites that act as docking sites for recruitment of additional proteins to form a signalling complex (Gibbins et al., 1998; Pasquet et al., 1999b; Sarkar, 1998). For example, PLCγ2 and PI3K (p85/p110) are brought to the vicinity of their substrates at the plasma membrane through interaction with tyrosine-phosphorylated LAT (Gibbins et al., 1998; Gross et al., 1999b).

PI3K generates phosphatidylinositol (3,4)-bisphosphate [PtdIns(3,4)P2] and phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] (Foster et al., 2003), which enable the recruitment to the plasma membrane of proteins that possess specific pleckstrin homology (PH) domains, including PLCγ2 and Btk, a tyrosine kinase that is also associated with Lyn (Pasquet et al., 1999b; Pasquet et al., 2000; Quek et al., 1998). Btk is believed to be partially responsible for the tyrosine phosphorylation of PLCγ2, which becomes activated and generates the second messengers inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] and diacylglycerol (DAG) (Oda et al., 2000; Quek et al., 1998). Ins(1,4,5)P3-mediated mobilization of calcium and DAG-mediated activation of PKC are essential components of the platelet activation process, irrespective of the agonist, and are necessary for platelet secretion and aggregation.

Several other proteins that appear to be important for the regulation of PLCγ2 are recruited to tyrosine-phosphorylated LAT. These include the Syk substrate SLP-76, the loss of which results in reduced tyrosine phosphorylation of PLCγ2 (Gross et al., 1999a; Gross et al., 1999b; Leo et al., 2002). Several adaptor proteins such as Gads, Grb2, Cbl and SLAP-130, and the GTP-exchange factor Vav, are also recruited to the signalling complex (Asazuma et al., 2000; Pearce et al., 2002). The specific roles of these molecules remain to be established, although Vav1-deficient (but not Vav2-deficient) mouse platelets display weakly diminished GPVI-stimulated aggregation responses (Pearce et al., 2002).

PI3K is important for platelet adhesion, spreading and aggregation (Falet et al., 2000; Pasquet et al., 1999a), activating several other signalling molecules, most notably protein kinase B (PKB, also known as Akt) (Barry and Gibbins, 2002; Kroner et al., 2000; Woulfe et al., 2004). Platelets possess two isoforms of PKB: PKBα and PKBβ (Akt1 and Akt2). PKBβ is important for normal platelet function and thrombus formation (Woulfe et al., 2004). PKBβ-null platelets have impaired alpha and dense granule secretion, and also impaired activation of integrin αIIbβ3. A downstream target of PKB is glycogen synthase kinase 3 (GSK3) (Doble and Woodgett, 2003), which is inactivated by phosphorylation (Barry et al., 2003). Since a range of GSK3 inhibitors inhibit platelet aggregation (Barry et al., 2003), GSK3 might have a negative regulatory function in platelets following stimulation. However, it should be noted that GSK3 might be phosphorylated by kinases other than PKB.

Another downstream effector of PI3K in platelets is integrin-linked kinase (ILK), a serine/threonine kinase that interacts with the cytoplasmic tails of β1 and β3 integrin subunits (Hannigan et al., 1996). ILK in platelets is probably important for both outside-in and inside-out signalling by the integrins α2β1 and αIIbβ3 (Pasquet et al., 2002; Stevens et al., 2004; Yamaji et al., 2002). It is speculated that ILK is responsible for the regulation of PKB by phosphorylating Ser473. The other regulatory site in PKB is Thr308, which is phosphorylated by phosphoinositide-dependent kinase 1 (PDK1). This enzyme is present in platelets and, upon platelet activation, forms a ternary complex with PKB and ILK (Barry and Gibbins, 2002).

Many of the early signalling events following stimulation of GPVI have been characterized. However, our knowledge of GPVI signalling is far from complete, and detail of events more distal from the receptor, such as PI3K-dependent signalling, is less well defined. An added layer of complexity is synergism between signalling mechanisms employed by adhesion receptors such as GPIb-V-IX and GPVI, in addition to synergism with secondary soluble agonists secreted from activated platelets. Although many questions remain to be answered, the culmination of platelet activation is inside-out signalling that upregulates the affinity of integrin αIIbβ3 and integrin α2β1, which facilitate thrombus formation.

Integrin signalling

Integrin αIIbβ3 also exhibits outside-in signalling upon ligation, which enhances the activation process through positive feedback. Integrin αIIbβ3 signalling has been extensively studied and is the subject of several recent reviews to which readers are directed (Calderwood, 2004; Calderwood et al., 2000; Eto et al., 2002; Liddington and Ginsberg, 2002; Phillips et al., 2001a; Phillips et al., 2001b; Tadokoro et al., 2003).

Research focusing on the stimulation of tyrosine kinase signalling in platelets in suspension indicated initially that integrin α2β1 does not engage in outside-in signalling (Hers et al., 2000). However, the use of recently developed α2β1-selective ligands indicates that this is incorrect. The adhesion of platelets to GFOGER peptides is accompanied by the tyrosine phosphorylation of several proteins, including Src, Syk, SLP-76 and PLCγ2, which are also involved in GPVI signalling, and calcium-dependent spreading (Inoue et al., 2003). p38 MAP kinase, ILK, Rac and PAK have also been implicated downstream of α2β1 ligation (Stevens et al., 2004; Sundaresan and Farndale, 2003; Suzuki-Inoue et al., 2001). A more detailed map combining these molecules into an α2β1 signalling pathway has yet to be established, and this is an important focus for future research.

Inhibitory platelet adhesion receptor signalling

Negative regulation of platelets is essential to prevent uncontrolled thrombosis. The roles of nitric oxide (NO) and prostacyclin (PGI2) are well established in the inhibition of platelet function (Geiger, 2001; Radomski et al., 1987). However, platelet activation can also be inhibited by signalling through the adhesion molecule PECAM-1 (also known as CD31) (Cicmil et al., 2002; Jones et al., 2001; Patil et al., 2001). PECAM-1 is expressed on several blood cell types and on endothelial cells, and is associated with the regulation of a range of processes, including trans-endothelial migration of leukocytes, regulation of cell activation and regulation of apoptosis (Newman and Newman, 2003). The functions of PECAM-1 are believed to be mediated by homophilic ligand binding (Albelda et al., 1991), although integrin αvβ3 and CD38 have also been proposed as ligands (Buckley et al., 1996). PECAM-1 dimerization has been shown to support adhesive properties of the molecule, and oligomerization causes cell signalling (Zhao and Newman, 2001).

The first evidence that PECAM-1 regulates the function of platelets in vivo was reported in 1994 in studies showing that time to vascular occlusion was increased following intravenous injection of anti-PECAM-1 antibodies in a mouse vascular injury model (Rosenblum et al., 1994). This was attributed to the inhibition by anti-PECAM-1 antibodies of PECAM-1-mediated platelet adhesion. More-recent studies indicate that this effect might have been due to the stimulation of PECAM-1 signalling. When stimulated through homophilic interactions and/or clustering, PECAM-1 is tyrosine phosphorylated on immunoreceptor tyrosine-based inhibition motifs [ITIMs; consensus: L/I/V/S-X-Y-X-X-L/V (Burshtyn et al., 1997)] in its cytoplasmic tail (Gibbins, 2002; Jackson et al., 1997a) by Src-family kinases. This facilitates the recruitment of tyrosine, serine/threonine or possibly lipid phosphatases, and the consequent inhibition of kinase-dependent signalling (Cicmil et al., 2000; Jackson et al., 1997b; Relou et al., 2003). The protein tyrosine phosphatases Shp-1 and Shp-2 and the serine/threonine protein phosphatase PP2A associate with PECAM-1 in platelets (Jackson et al., 1997b; Relou et al., 2003). Although PECAM-1 signalling reduces total platelet protein tyrosine phosphorylation, inositol phosphate production, calcium mobilization and PI3K signalling (Cicmil et al., 2002; Jones et al., 2001; Thai et al., 2003), specific substrates for these phosphatases in platelets are currently unclear.

PECAM-1 also inhibits GPIb-mediated platelet activation (Rathore et al., 2003) and downregulates FcγRIIA-mediated platelet responses (Thai et al., 2003). The effects of PECAM-1 appear not to be restricted to inhibition of ITAM-mediated signalling: thrombin-dependent and oxidized low-density lipoprotein (LDL)-stimulated platelet signalling are also inhibited (Cicmil et al., 2002; Relou et al., 2003).

Platelet PECAM-1 becomes tyrosine phosphorylated following stimulation of platelets with a range of agonists and upon platelet aggregation, suggesting a negative-feedback role (Cicmil et al., 2000; Jones et al., 2001). Since the principal ligand for PECAM-1 is PECAM-1 itself (Albelda et al., 1991), interactions between PECAM-1 on platelets and endothelial cells might restrict the growth of a thrombus through the feedback mechanisms described above. Indeed, thrombi formed in PECAM-1-null mice are larger and more stable in comparison with those formed in wild-type mice (Falati et al., 2003). The balance between signalling through activatory adhesion receptors and receptors for soluble platelet agonists, and signalling stimulated by PECAM-1 may regulate the stimulus threshold for thrombus formation and may determine thrombus size and stability.

Perspective

Our understanding of the receptors and signalling mechanisms that regulate thrombus formation has advanced markedly in recent years. The ability of platelets to respond specifically and rapidly to subendothelial proteins exposed upon tissue injury, and under conditions of shear stress, is crucial for effective haemostasis. The identification and characterization of the platelet adhesion receptors GPIb-V-IX and GPVI, and integrins αIIbβ3 and α2β1, are important milestones in our understanding of these processes. The complementary roles of these receptors in platelet adhesion and cell signalling leading to thrombus formation are clearly established. Many of the early signalling events following GPVI ligation are characterized, although our understanding of GPIb-V-IX is less advanced. With many of the spectrum of molecules involved in GPVI and integrin αIIbβ3 signalling also implicated in GPIb-V-IX signalling, rapid progress is anticipated in this area. Similarly, our knowledge of the signalling mechanisms through which PECAM-1 inhibits activatory signals from receptors such as GPVI and GPIb-V-IX lacks depth, and this too is an important area for future development.

The biggest challenge presented to researchers studying platelet biology is to relate the significance of platelet signalling and function in vitro to the in vivo situation of haemostasis and thrombosis. The use of in vivo models of thrombosis, as well as sophisticated methodology to measure platelet signalling and thrombus formation under flow, are important technical developments towards this aim.

The translation of basic research toward new strategies to prevent arterial thrombosis underscores much of the research in this area. New avenues for investigation are presented by the potential benefits of blocking the interactions of VWF with GPIb-V-IX, and collagen with GPVI and/or integrin α2β1, or the activation of PECAM-1, or pharmacological regulation of the signalling mechanisms employed by these receptors. A substantial challenge is the targeting of pathological thrombi yet with minimal side-effects (e.g. haemorrhage). The platelet-specific expression of GPIb-V-IX and GPVI, and the well-tolerated deletion of the gene encoding GPVI in mice, indicate these molecules may underlie future advances in anti-platelet therapy.

Acknowledgements

We acknowledge valuable discussions with Dr A. Poole (University of Bristol, UK) during the preparation of this manuscript. Research in the author's laboratory is supported by grants from the Medical Research Council, the Biotechnology and Biological Sciences Research Council, the British Heart Foundation and the Wellcome Trust.

References

Albelda, S. M., Muller, W. A., Buck, C. A. and Newman, P. J. (
1991
). Molecular and cellular properties of PECAM-1 (endoCAM/CD31): a novel vascular cell-cell adhesion molecule.
J. Cell Biol.
114
,
1059
-1068.
Alevriadou, B. R., Moake, J. L., Turner, N. A., Ruggeri, Z. M., Folie, B. J., Phillips, M. D., Schreiber, A. B., Hrinda, M. E. and McIntire, L. V. (
1993
). Real-time analysis of shear-dependent thrombus formation and its blockade by inhibitors of von Willebrand-factor binding to platelets.
Blood
81
,
1263
-1276.
Andrews, R. K., Harris, S. J., McNally, T. and Berndt, M. C. (
1998
). Binding of purified 14-3-3 zeta signaling protein to discrete amino acid sequences within the cytoplasmic domain of the platelet membrane glycoprotein Ib-IX-V complex.
Biochemistry
37
,
638
-647.
Andrews, R. K., Munday, A. D., Mitchell, C. A. and Berndt, M. C. (
2001
). Interaction of calmodulin with the cytoplasmic domain of the platelet membrane glycoprotein Ib-IX-V complex.
Blood
98
,
681
-687.
Arai, M., Yamamoto, N., Moroi, M., Akamatsu, N., Fukutake, K. and Tanoue, K. (
1995
). Platelets with 10% of the normal amount of glycoprotein VI have an impaired response to collagen that results in a mild bleeding tendency.
Br. J. Haematol.
89
,
124
-130.
Asazuma, N., Ozaki, Y., Satoh, K., Yatomi, Y., Handa, M., Fujimura, Y., Miura, S. and Kume, S. (
1997
). Glycoprotein Ib von Willebrand factor interactions activate tyrosine kinases in human platelets.
Blood
90
,
4789
-4798.
Asazuma, N., Wilde, J. I., Berlanga, O., Leduc, M., Leo, A., Schweighoffer, E., Tybulewicz, V., Bon, C., Liu, S. K., McGlade, C. J. et al. (
2000
). Interaction of linker for activation of T cells with multiple adapter proteins in platelets activated by the glycoprotein VI-selective ligand, convulxin.
J. Biol. Chem.
275
,
33427
-33434.
Asselin, J., Gibbins, J. M., Achison, M., Lee, Y. H., Morton, L. F., Farndale, R. W., Barnes, M. J. and Watson, S. P. (
1997
). A collagen-like peptide stimulates tyrosine phosphorylation of syk and phospholipase Cγ2 in platelets independent of the integrin α2β1.
Blood
89
,
1235
-1242.
Barnes, M. J. and Farndale, R. W. (
1999
). Collagens and atherosclerosis.
Exp. Gerontol.
34
,
513
-525.
Barry, F. A. and Gibbins, J. M. (
2002
). Protein kinase B is regulated in platelets by the collagen receptor glycoprotein VI.
J. Biol. Chem.
277
,
12874
-12878.
Barry, F. A., Graham, G. J., Fry, M. J. and Gibbins, J. M. (
2003
). Regulation of glycogen synthase kinase 3 in human platelets: a possible role in platelet function?
FEBS Lett.
553
,
173
-178.
Batuwangala, T., Leduc, M., Gibbins, J. M., Bon, C. and Jones, E. Y. (
2004
). Structure of the snake-venom toxin convulxin.
Acta Crystallogr. D Biol. Crystallogr.
60
,
46
-53.
Benhamou, M., Ryba, N. J., Kihara, H., Nishikata, H. and Siraganian, R. P. (
1993
). Protein-tyrosine kinase p72syk in high affinity IgE receptor signaling. Identification as a component of pp72 and association with the receptor gamma chain after receptor aggregation.
J. Biol. Chem.
268
,
23318
-23324.
Blank, U., Ra, C., Miller, L., White, K., Metzger, H. and Kinet, J. P. (
1989
). Complete structure and expression in transfected cells of high affinity IgE receptor.
Nature
337
,
187
-189.
Briddon, S. J. and Watson, S. P. (
1999
). Evidence for the involvement of p59(fyn) and p53/56(lyn) in collagen receptor signalling in human platelets.
Biochem. J.
338
,
203
-209.
Buckley, C. D., Doyonnas, R., Newton, J. P., Blystone, S. D., Brown, E. J., Watt, S. M. and Simmons, D. L. (
1996
). Identification of αvβ3 as a heterotypic ligand for CD31/PECAM-1.
J. Cell Sci.
109
,
437
-445.
Burshtyn, D. N., Yang, W. T., Yi, T. L. and Long, E. O. (
1997
). A novel phosphotyrosine motif with a critical amino acid at position-2 for the SH2 domain-mediated activation of the tyrosine phosphatase SHP-1.
J. Biol. Chem.
272
,
13066
-13072.
Calderwood, D. A. (
2004
). Integrin activation.
J. Cell Sci.
117
,
657
-666.
Calderwood, D. A., Shattil, S. J. and Ginsberg, M. H. (
2000
). Integrins and actin filaments: reciprocal regulation of cell adhesion and signaling.
J. Biol. Chem.
275
,
22607
-22610.
Canobbio, I., Bertoni, A., Lova, P., Paganini, S., Hirsch, E., Sinigaglia, F., Balduini, C. and Torti, M. (
2001
). Platelet activation by von Willebrand Factor requires coordinated signaling through thromboxane A(2) and Fc gamma IIA receptor.
J. Biol. Chem.
276
,
26022
-26029.
Chen, J. C., Diacovo, T. G., Grenache, D. G., Santoro, S. A. and Zutter, M. M. (
2002
). The alpha(2) integrin subunit-deficient mouse – a multifaceted phenotype including defects of branching morphogenesis and hemostasis.
Am. J. Pathol.
161
,
337
-344.
Chiang, T., Rinaldy, A. and Kang, A. (
1997
). Cloning, characterization, and functional studies of a nonintegrin platelet receptor for type I collagen.
J. Clin. Invest.
100
,
514
-521.
Chiang, T. M., Cole, F. and Woo-Rasberry, V. (
2002
). Cloning, characterization, and functional studies of a 47-kDa platelet receptor for type III collagen.
J. Biol. Chem.
277
,
34896
-34901.
Cicmil, M., Thomas, J. M., Sage, T., Barry, F. A., Leduc, M., Bon, C. and Gibbins, J. M. (
2000
). Collagen, convulxin, and thrombin stimulate aggregation-independent tyrosine phosphorylation of CD31 in platelets. Evidence for the involvement of Src family kinases.
J. Biol. Chem.
275
,
27339
-27347.
Cicmil, M., Thomas, J. M., Leduc, M., Bon, C. and Gibbins, J. M. (
2002
). Platelet endothelial cell adhesion molecule-1 signaling inhibits the activation of human platelets.
Blood
99
,
137
-144.
Clemetson, J. M., Polgar, J., Magnenat, E., Wells, T. N. C. and Clemetson, K. J. (
1999
). The platelet collagen receptor glycoprotein VI is a member of the immunoglobulin superfamily closely related to Fc alpha R and the natural killer receptors.
J. Biol. Chem.
274
,
29019
-29024.
Cunningham, J. G., Meyer, S. C. and Fox, J. E. B. (
1996
). The cytoplasmic domain of the alpha-subunit of glycoprotein (GP) Ib mediates attachment of the entire GP Ib-IX complex to the cytoskeleton and regulates von Willebrand factor-induced changes in cell morphology.
J. Biol. Chem.
271
,
11581
-11587.
Daniel, J. L., Dangelmaier, C., Strouse, R. and Smith, J. B. (
1994
). Collagen induces normal signal transduction in platelets deficient in CD36 (platelet glycoprotein IV).
Thromb. Haemost.
71
,
353
-356.
Doble, B. W. and Woodgett, J. R. (
2003
). GSK-3: tricks of the trade for a multi-tasking kinase.
J. Cell Sci.
116
,
1175
-1186.
Eto, K., Murphy, R., Kerrigan, S. W., Bertoni, A., Stuhlmann, H., Nakano, T., Leavitt, A. D. and Shattil, S. J. (
2002
). Megakaryocytes derived from embryonic stem cells implicate CalDAG-GEFI in integrin signaling.
Proc. Natl. Acad. Sci. USA
99
,
12819
-12824.
Ezumi, Y., Shindoh, K., Tsuji, M. and Takayama, H. (
1998
). Physical and functional association of the src family kinases fyn and lyn with the collagen receptor glycoprotein VI-Fc receptor gamma chain complex on human platelets.
J. Exp. Med.
188
,
267
-276.
Ezumi, Y., Uchiyama, T. and Takayama, H. (
2000
). Molecular cloning, genomic structure, chromosomal localization, and alternative splice forms of the platelet collagen receptor glycoprotein VI.
Biochem. Biophys. Res. Commun.
277
,
27
-36.
Ezumi, Y., Kodama, K., Uchiyama, T. and Takayama, H. (
2002
). Constitutive and functional association of the platelet collagen receptor glycoprotein VI-Fc receptor gamma-chain complex with membrane rafts.
Blood
99
,
3250
-3255.
Falati, S., Edmead, C. E. and Poole, A. W. (
1999
). Glycoprotein Ib-V-IX, a receptor for von Willebrand factor, couples physically and functionally to the Fc receptor gamma-chain, Fyn, and Lyn to activate human platelets.
Blood
94
,
1648
-1656.
Falati, S., Gross, P., Merrill-Skoloff, G., Furie, B. C. and Furie, B. (
2002
). Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse.
Nat. Med.
8
,
1175
-1180.
Falati, S., Patil, S., Gibbins, J., Gross, P. L., Merrill-Skoloff, G., Weiler, H., Cooley, B., Newman, D. K., Newman, P. J., Furie, B. C. et al. (
2003
). Opposing effects of P-selectin and PECAM-1 on arterial thrombus growth and stability in vivo.
J. Thromb. Haemost. Suppl. 1
, OC054.
Falet, H., Barkalow, K. L., Pivniouk, V. I., Barnes, M. J., Geha, R. S. and Hartwig, J. H. (
2000
). Roles of SLP-76, phosphoinositide 3-kinase, and gelsolin in the platelet shape changes initiated by the collagen receptor GPVI/FcR gamma-chain complex.
Blood
96
,
3786
-3792.
Farndale, R. W., Sixma, J. J., Barnes, M. J. and de Groot, P. G. (
2004
). The role of collagen in thrombosis and hemostasis.
J. Thromb. Haemost.
2
,
561
-573.
Foster, F. M., Traer, C. J., Abraham, S. M. and Fry, M. J. (
2003
). The phosphoinositide (PI) 3-kinase family.
J. Cell Sci.
116
,
3037
-3040.
Francischetti, I. M. B., Saliou, B., Leduc, M., Carlini, C. R., Hatmi, M., Randon, J., Faili, A. and Bon, C. (
1997
). Convulxin, a potent platelet-aggregating protein from Crotalus durissus terrificus venom, specifically binds to platelets.
TOXICON
35
,
1217
-1228.
Geiger, J. (
2001
). Inhibitors of platelet signal transduction as anti-aggregatory drugs.
Expert Opin. Investig. Drugs
10
,
865
-890.
Gibbins, J. M. (
2002
). The negative regulation of platelet function: extending the role of the ITIM.
Trends Cardiovasc. Med.
12
,
213
-219.
Gibbins, J., Asselin, J., Farndale, R., Barnes, M., Law, C. L. and Watson, S. P. (
1996
). Tyrosine phosphorylation of the Fc receptor gamma-chain in collagen-stimulated platelets.
J. Biol. Chem.
271
,
18095
-18099.
Gibbins, J. M., Okuma, M., Farndale, R., Barnes, M. and Watson, S. P. (
1997
). Glycoprotein VI is the collagen receptor in platelets which underlies tyrosine phosphorylation of the Fc receptor γ-chain.
FEBS Lett.
413
,
255
-259.
Gibbins, J. M., Briddon, S., Shutes, A., van Vugt, M. J., de Winkel, J. G. J., Saito, T. and Watson, S. P. (
1998
). The p85 subunit of phosphatidylinositol 3-kinase associates with the Fc receptor gamma-chain and linker for activator of T cells (LAT) in platelets stimulated by collagen and convulxin.
J. Biol. Chem.
273
,
34437
-34443.
Gross, B. S., Lee, J. R., Clements, J. L., Turner, M., Tybulewicz, V. L. J., Findell, P. R., Koretzky, G. A. and Watson, S. P. (
1999a
). Tyrosine phosphorylation of SLP-76 is downstream of Syk following stimulation of the collagen receptor in platelets.
J. Biol. Chem.
274
,
5963
-5971.
Gross, B. S., Melford, S. K. and Watson, S. P. (
1999b
). Evidence that phospholipase C-γ2 interacts with SLP-76, syk, lyn, LAT and the Fc receptor γ-chain after stimulation of the collagen receptor glycoprotein VI in human platelets.
Eur. J. Biochem.
263
,
612
-623.
Hannigan, G. E., Leung-Hagesteijn, C., Fitz-Gibbon, L., Coppolino, M. G., Radeva, G., Filmus, J., Bell, J. C. and Dedhar, S. (
1996
). Regulation of cell adhesion and anchorage-dependent growth by a new β1-integrin-linked protein kinase.
Nature
379
,
91
-96.
Hartwig, J. and Italiano, J. (
2003
). The birth of the platelet.
J. Thromb. Haemost.
1
,
1580
-1586.
Hashimoto, T., Wakabayashi, T., Watanabe, A., Kowa, H., Hosoda, R., Nakamura, A., Kanazawa, I., Arai, T., Takio, K., Mann, D. M. A. et al. (
2002
). CLAC: a novel Alzheimer amyloid plaque component derived from a transmembrane precursor, CLAC-P/collagen type XXV.
EMBO J.
21
,
1524
-1534.
Heemskerk, J. W. M., Bevers, E. M. and Lindhout, T. (
2002
). Platelet activation and blood coagulation.
Thromb. Haemost.
88
,
186
-193.
Hers, I., Berlanga, O., Tiekstra, M. J., Kamiguti, A. S., Theakston, R. D. G. and Watson, S. P. (
2000
). Evidence against a direct role of the integrin alpha 2 beta 1 in collagen-induced tyrosine phosphorylation in human platelets.
Eur. J. Biochem.
267
,
2088
-2097.
Holtkotter, O., Nieswandt, B., Smyth, N., Muller, M., Hafner, M., Schulte, V., Krieg, T. and Eckes, B. (
2002
). Integrin alpha(2)-deficient mice develop normally, are fertile, but display partially defective platelet interaction with collagen.
J. Biol. Chem.
277
,
10789
-10794.
Inoue, O., Suzuki-Inoue, K., Dean, W. L., Frampton, J. and Watson, S. P. (
2003
). Integrin alpha(2)beta(1) mediates outside-in regulation of platelet spreading on collagen through activation of Src kinases and PLC-gamma 2.
J. Cell Biol.
160
,
769
-780.
Italiano, J. E., Lecine, P., Shivdasani, R. A. and Hartwig, J. H. (
1999
). Blood platelets are assembled principally at the ends of proplatelet processes produced by differentiated megakaryocytes.
J. Cell Biol.
147
,
1299
-1312.
Jackson, D. E., Kupcho, K. R. and Newman, P. J. (
1997a
). Characterization of phosphotyrosine binding motifs in the cytoplasmic domain of platelet/endothelial cell adhesion molecule-1 (PECAM-1) that are required for the cellular association and activation of the protein-tyrosine phosphatase, SHP-2.
J. Biol. Chem.
272
,
24868
-24875.
Jackson, D. E., Ward, C. M., Wang, R. and Newman, P. J. (
1997b
). The protein-tyrosine phosphatase SHP-2 binds platelet/endothelial cell adhesion molecule-1 (PECAM-1) and forms a distinct signaling complex during platelet aggregation.
J. Biol. Chem.
272
,
6986
-6993.
Jackson, S. P., Schoenwaelder, S. M., Yuan, Y. P., Rabinowitz, I., Salem, H. H. and Mitchell, C. A. (
1994
). Adhesion receptor activation of phosphatidylinositol 3-kinase – Von-Willebrand-factor stimulates the cytoskeletal association and activation of phosphatidylinositol 3-kinase and Pp60(C-Src) in human platelets.
J. Biol. Chem.
269
,
27093
-27099.
Jackson, S. P., Nesbitt, W. S. and Kulkarni, S. (
2003
). Signaling events underlying thrombus formation.
J. Thromb. Haemost.
1
,
1602
-1612.
Jandrot-Perrus, M., Busfield, S., Lagrue, A. H., Xiong, X. M., Debili, N., Chickering, T., le Couedic, J. P., Goodearl, A., Dussault, B., Fraser, C. et al. (
2000
). Cloning, characterization, and functional studies of human and mouse glycoprotein VI: a platelet-specific collagen receptor from the immunoglobulin superfamily.
Blood
96
,
1798
-1807.
Jones, K. L., Hughan, S. C., Dopheide, S. M., Farndale, R. W., Jackson, S. P. and Jackson, D. E. (
2001
). Platelet endothelial cell adhesion molecule-1 is a negative regulator of platelet-collagen interactions.
Blood
98
,
1456
-1463.
Jung, S. M. and Moroi, M. (
1998
). Platelets interact with soluble and insoluble collagens through characteristically different reactions.
J. Biol. Chem.
273
,
14827
-14837.
Jung, S. M. and Moroi, M. (
2000
). Signal-transducing mechanisms involved in activation of the platelet collagen receptor integrin alpha(2)beta(1).
J. Biol. Chem.
275
,
8016
-8026.
Kahn, M. L., Diacovo, T. G., Bainton, D. F., Lanza, F., Trejo, J. and Coughlin, S. (
1999
). Glycoprotein V-deficient platelets have undiminished thrombin responsiveness and do not exhibit a Bernard-Soulier phenotype.
Blood
94
,
4112
-4121.
Kanaji, S., Kanaji, T., Furihata, K., Kato, K., Ware, J. L. and Kunicki, T. J. (
2003
). Convulxin binds to native, human glycoprotein Ib alpha.
J. Biol. Chem.
278
,
39452
-39460.
Kasirer-Friede, A., Cozzi, M. R., Mazzucato, M., de Marco, L., Ruggeri, Z. M. and Shattil, S. J. (
2004
). Signalling through GP Ib-IX-V activates αIIbβ3 independently of other receptors.
Blood
103
,
3403
-3411.
Kato, K., Kanaji, T., Russell, S., Kunicki, T. J., Furihata, K., Kanaji, S., Marchese, P., Reininger, A., Ruggeri, Z. M. and Ware, J. (
2003
). The contribution of glycoprotein VI to stable platelet adhesion and thrombus formation illustrated by targeted gene deletion.
Blood
102
,
1701
-1707.
Kehrel, B., Wierwille, S., Clemetson, K. J., Anders, O., Steiner, M., Knight, C. G., Farndale, R., Okuma, M. and Barnes, M. J. (
1998
). Glycoprotein VI is a major collagen receptor for platelet activation: it recognizes the platelet-activating quaternary structure of collagen, whereas CD36, glycoprotein IIb/IIIa, and von Willibrand factor do not.
Blood
91
,
491
-499.
Knight, C. G., Morton, L. F., Onley, D. J., Peachey, A. R., Messent, A. J., Smethurst, P. A., Tuckwell, D. S., Farndale, R. W. and Barnes, M. J. (
1998
). Identification in collagen type I of an integrin α2β1-binding site containing an essential GER sequence.
J. Biol. Chem.
273
,
33287
-33294.
Kroll, M. H., Harris, T. S., Moake, J. L., Handin, R. I. and Schafer, A. I. (
1991
). von Willebrand-factor binding to platelet GpIb initiates signals for platelet activation.
J. Clin. Invest.
88
,
1568
-1573.
Kroner, C., Eybrechts, K. and Akkerman, J. W. N. (
2000
). Dual regulation of platelet protein kinase B.
J. Biol. Chem.
275
,
27790
-27798.
Kuijpers, M. J. E., Schulte, V., Bergmeier, W., Lindhout, T., Brakebusch, C., Offermanns, S., Fassler, R., Heemskerk, J. W. M. and Nieswandt, B. (
2003
). Complementary roles of platelet glycoprotein VI and integrin alpha 2 beta 1 in collagen-induced thrombus formation in flowing whole blood ex vivo.
FASEB J.
17
,
U372
-U394.
Kunicki, T. J., Nugent, D. J., Staats, S. J., Orchekowski, R. P., Wayner, E. A. and Carter, W. G. (
1988
). The human fibroblast class-Ii extracellular-matrix receptor mediates platelet-adhesion to collagen and is identical to the platelet glycoprotein-Ia-Iia complex.
J. Biol. Chem.
263
,
4516
-4519.
Kuster, H., Thompson, H. and Kinet, J. P. (
1990
). Characterization and expression of the gene for the human Fc receptor γ subunit.
J. Biol. Chem.
265
,
6448
-6452.
Kuwahara, M., Sugimoto, M., Tsuji, S., Miyata, S. and Yoshioka, A. (
1999
). Cytosolic calcium changes in a process of platelet adhesion and cohesion on a von Willebrand factor-coated surface under flow conditions.
Blood
94
,
1149
-1155.
Leo, L., di Paola, J., Judd, B. A., Koretzky, G. A. and Lentz, S. R. (
2002
). Role of the adapter protein SLP-76 in GPVI-dependent platelet procoagulant responses to collagen.
Blood
100
,
2839
-2844.
Li, Z. Y., Xi, X. D., Gu, M. Y., Feil, R., Ye, R. D., Eigenthaler, M., Hofmann, F. and Du, X. P. (
2003
). A stimulatory role for cGMP-dependent protein kinase in platelet activation.
Cell
112
,
77
-86.
Liddington, R. C. and Ginsberg, M. H. (
2002
). Integrin activation takes shape.
J. Cell Biol.
158
,
833
-839.
Locke, D., Chen, H., Liu, Y., Liu, C. D. and Kahn, M. L. (
2002
). Lipid rafts orchestrate signaling by the platelet receptor glycoprotein VI.
J. Biol. Chem.
277
,
18801
-18809.
Marshall, S. J., Asazuma, N., Best, D., Wonerow, P., Salmon, G., Andrews, R. K. and Watson, S. P. (
2002
). Glycoprotein IIb-IIIa-dependent aggregation by glycoprotein Ib alpha is reinforced by a Src family kinase inhibitor (PP1)-sensitive signalling pathway.
Biochem. J.
361
,
297
-305.
Massberg, S., Gawaz, M., Gruner, S., Schulte, V., Konrad, I., Zohlnhofer, D., Heinzmann, U. and Nieswandt, B. (
2003
). A crucial role of glycoprotein VI for platelet recruitment to the injured arterial wall in vivo.
J. Exp. Med.
197
,
41
-49.
Mazzucato, M., Pradella, P., Cozzi, M. R., de Marco, L. and Ruggeri, Z. M. (
2002
). Sequential cytoplasmic calcium signals in a 2-stage platelet activation process induced by the glycoprotein Ib alpha mechanoreceptor.
Blood
100
,
2793
-2800.
Milner, E. P., Zheng, Q. and Kermode, J. C. (
1998
). Ristocetin-mediated interaction of human von Willebrand factor with platelet glycoprotein Ib evokes a transient calcium signal: observations with Fura-PE3.
J. Lab. Clin. Med.
131
,
49
-62.
Moake, J. L., Turner, N. A., Stathopoulos, N. A., Nolasco, L. and Hellums, J. D. (
1988
). Shear-induced platelet-aggregation can be mediated by Vwf released from platelets, as well as by exogenous large or unusually large Vwf multimers, requires adenosine-diphosphate, and is resistant to aspirin.
Blood
71
,
1366
-1374.
Monnet, E. and Fauvel-Lefeve, F. (
2000
). A new platelet receptor specific to type III collagen. Type III collagen-binding protein.
J. Biol. Chem.
275
,
10912
-10917.
Monnet, E., Depraetere, H., Legrand, C., Deckmyn, H. and Fauvel-Lafeve, F. (
2001
). A monoclonal antibody to platelet type III collagen-binding protein (TIIICBP) binds to blood and vascular cells, and inhibits platelet vessel-wall interactions.
Thromb. Haemost.
86
,
694
-701.
Moroi, M., Jung, S. M., Okuma, M. and Shinmyozu, K. (
1989
). A patient with platelets deficient in glycoprotein VI that lack both collagen-induced aggregation and adhesion.
J. Clin. Invest.
84
,
1440
-1445.
Moroi, M., Jung, S. M., Shinmyozu, K., Tomiyama, Y., Ordinas, A. and Diaz-Ricart, M. (
1996
). Analysis of platelet adhesion to a collagen-coated surface under flow conditions: The involvement of glycoprotein VI in the platelet adhesion.
Blood
88
,
2081
-2092.
Moroi, M., Onitsuka, I., Imaizumi, T. and Jung, S. M. (
2000
). Involvement of activated integrin alpha(2)beta(1) in the firm adhesion of platelets onto a surface of immobilized collagen under flow conditions.
Thromb. Haemost.
83
,
769
-776.
Morton, L. F., Peachey, A. R. and Barnes, M. J. (
1989
). Platelet-reactive sites in collagens type I and type III. Evidence for separate adhesion and aggregatory sites.
Biochem. J.
258
,
157
-163.
Morton, L. F., Peachey, A. R., Zijenah, L. S., Goodall, A. H. and Humphries, M. J. (
1994
). Conformation-dependent platelet adhesion to collagen involving integrin α2β1-mediated and other mechanisms: multiple α2β1-recognition sites in collagen type I.
Biochem. J.
299
,
791
-797.
Morton, L. F., Hargreaves, P. G., Farndale, R. W., Young, R. D. and Barnes, M. J. (
1995
). Integrin alpha 2 beta 1-independent activation of platelets by simple collagen-like peptides: collagen tertiary (triple-helical) and quaternary (polymeric) structures are sufficient alone for alpha 2 beta 1-independent platelet reactivity.
Biochem. J.
306
,
337
-344.
Munday, A. D., Berndt, M. C. and Mitchell, C. A. (
2000
). Phosphoinositide 3-kinase forms a complex with platelet membrane glycoprotein Ib-IX-V complex and 14-3-3 zeta.
Blood
96
,
577
-584.
Nesbitt, W. S., Kulkarni, S., Giuliano, S., Goncalves, I., Dopheide, S. M., Yap, C. L., Harper, I. S., Salem, H. H. and Jackson, S. P. (
2002
). Distinct glycoprotein Ib/V/IX and integrin alpha(IIb)beta(3)-dependent calcium signals cooperatively regulate platelet adhesion under flow.
J. Biol. Chem.
277
,
2965
-2972.
Newman, P. J. and Newman, D. K. (
2003
). Signal transduction pathways mediated by PECAM-1: new roles for an old molecule in platelet and vascular cell biology.
Arterioscler. Thromb. Vasc. Biol.
23
,
953
-964.
Nieswandt, B. and Watson, S. P. (
2003
). Platelet-collagen interaction: is GPVI the central receptor?
Blood
102
,
449
-461.
Nieswandt, B., Bergmeier, W., Schulte, V., Rackebrandt, K., Gessner, J. E. and Zirngibl, H. (
2000
). Expression and function of the mouse collagen receptor glycoprotein VI is strictly dependent on its association with the FcR gamma chain.
J. Biol. Chem.
275
,
23998
-24002.
Nieswandt, B., Brakebusch, C., Bergmeier, W., Schulte, V., Bouvard, D., Mokhtari-Nejad, R., Lindhout, T., Heemskerk, J. W. M., Zirngibl, H. and Fassler, R. (
2001a
). Glycoprotein VI but not alpha 2 beta 1 integrin is essential for platelet interaction with collagen.
EMBO J.
20
,
2120
-2130.
Nieswandt, B., Schulte, V., Bergmeier, W., Mokhtari-Nejad, R., Rackebrandt, K., Cazenave, J. P., Ohlmann, P., Gachet, C. and Zirngibl, H. (
2001b
). Long-term antithrombotic protection by in vivo depletion of platelet glycoprotein VI in mice.
J. Exp. Med.
193
,
459
-469.
Nieuwenhuis, H. K., Akkerman, J. W. N., Houdijk, W. P. M. and Sixma, J. J. (
1985
). Human-blood platelets showing no response to collagen fail to express surface glycoprotein-Ia.
Nature
318
,
470
-472.
Oda, A., Ikeda, Y., Ochs, H. D., Druker, B. J., Ozaki, K., Handa, M., Ariga, T., Sakiyama, Y., Witte, O. N. and Wahl, M. I. (
2000
). Rapid tyrosine phosphorylation and activation of Bruton's tyrosine/Tec kinases in platelets induced by collagen binding or CD32 cross-linking.
Blood
95
,
1663
-1670.
Ozaki, Y., Satoh, K., Yatomi, Y., Miura, S., Fujimura, Y. and Kume, S. (
1995
). Protein-tyrosine phosphorylation in human platelets induced by interaction between glycoprotein Ib and Von-Willebrand-factor.
Biochim. Biophys. Acta
1243
,
482
-488.
Pasquet, J. M., Bobe, R., Gross, B., Gratacap, M. P., Tomlinson, M. G., Payrastre, B. and Watson, S. P. (
1999a
). A collagen-related peptide regulates phospholipase Cgamma2 via phosphatidylinositol 3-kinase in human platelets.
Biochem. J.
342
,
171
-177.
Pasquet, J. M., Gross, B., Quek, L., Asazuma, N., Zhang, W. G., Sommers, C. L., Schweighoffer, E., Tybulewicz, V., Judd, B., Lee, J. R. et al. (
1999b
). LAT is required for tyrosine phosphorylation of phospholipase C gamma 2 and platelet activation by the collagen receptor GPVI.
Mol. Cell. Biol.
19
,
8326
-8334.
Pasquet, J. M., Quek, L., Stevens, C., Bobe, R., Huber, M., Duronio, V., Krystal, G. and Watson, S. P. (
2000
). Phosphatidylinositol 3,4,5-trisphosphate regulates Ca2+ entry via Btk in platelets and megakaryocytes without increasing phospholipase C activity.
EMBO J.
19
,
2793
-2802.
Pasquet, J. M., Noury, M. and Nurden, A. T. (
2002
). Evidence that the platelet integrin alpha IIb beta 3 is regulated by the integrin-linked kinase, ILK, in a PI3-kinase dependent pathway.
Thromb. Haemost.
88
,
115
-122.
Patil, S., Newman, D. K. and Newman, P. J. (
2001
). Platelet endothelial cell adhesion molecule-1 serves as an inhibitory receptor that modulates platelet responses to collagen.
Blood
97
,
1727
-1732.
Pearce, A. C., Wilde, J. I., Doody, G. M., Best, D., Inoue, O., Vigorito, E., Tybulewicz, V. L. J., Turner, M. and Watson, S. P. (
2002
). Vav1, but not Vav2, contributes to platelet aggregation by CRP and thrombin, but neither is required for regulation of phospholipase C.
Blood
100
,
3561
-3569.
Phillips, D. R., Nannizzi-Alamio, L. and Prasad, K. S. S. (
2001a
). beta 3 tyrosine phosphorylation in alpha IIb beta 3 (platelet membrane GP IIb-IIIa) outside-in integrin signaling.
Thromb. Haemost.
86
,
246
-258.
Phillips, D. R., Prasad, K. S. S., Manganello, J., Bao, M. and Nannizzi-Alaimo, L. (
2001b
). Integrin tyrosine phosphorylation in platelet signaling.
Curr. Opin. Cell Biol.
13
,
546
-554.
Polanowska-Grabowska, R. and Gear, A. R. L. (
1992
). High-speed platelet-adhesion under conditions of rapid flow.
Proc. Natl. Acad. Sci. USA
89
,
5754
-5758.
Poole, A., Gibbins, J. M., Turner, M., van Vugt, M. J., vandeWinkel, J. G. J., Saito, T., Tybulewicz, V. L. J. and Watson, S. P. (
1997
). The Fc receptor gamma-chain and the tyrosine kinase Syk are essential for activation of mouse platelets by collagen.
EMBO J.
16
,
2333
-2341.
Quek, L. S., Bolen, J. and Watson, S. P. (
1998
). A role for Bruton's tyrosine kinase (Btk) in platlet activation by collagen.
Curr. Biol.
8
,
1137
-1140.
Quek, L. S., Pasquet, J. M., Hers, I., Cornall, R., Knight, G., Barnes, M., Hibbs, M. L., Dunn, A. R., Lowell, C. A. and Watson, S. P. (
2000
). Fyn and Lyn phosphorylate the Fc receptor gamma chain downstream of glycoprotein VI in murine platelets, and Lyn regulates a novel feedback pathway.
Blood
96
,
4246
-4253.
Radomski, M. W., Palmer, R. M. J. and Moncada, S. (
1987
). Endogenous nitric-oxide inhibits human-platelet adhesion to vascular endothelium.
Lancet
2
,
1057
-1058.
Ramakrishnan, V., Reeves, P. S., DeGuzman, F., Deshpande, U., Ministri-Madrid, K., DuBridge, R. B. and Phillips, D. R. (
1999
). Increased thrombin responsiveness in platelets from mice lacking glycoprotein V.
Proc. Natl. Acad. Sci. USA
96
,
13336
-13341.
Rathore, V., Stapleton, M. A., Hillery, C. A., Montgomery, R. R., Nichols, T. C., Merricks, E. P., Newman, D. K. and Newman, P. J. (
2003
). PECAM-1 negatively regulates GPIb/V/IX signaling in murine platelets.
Blood
102
,
3658
-3664.
Razdan, K., Hellums, J. D. and Kroll, M. H. (
1994
). Shear-stress-induced Von-Willebrand-factor binding to platelets causes the activation of tyrosine kinases.
Biochem. J.
302
,
681
-686.
Relou, I. A. M., Gorter, G., Ferreira, I. A., van Rijn, H. J. M. and Akkerman, J. W. N. (
2003
). Platelet endothelial cell adhesion molecule-1 (PECAM-1) inhibits low density lipoprotein-induced signaling in platelets.
J. Biol. Chem.
278
,
32638
-32644.
Reth, M. (
1989
). Antigen receptor tail clue.
Nature
338
,
383
-384.
Rosenblum, W. I., Murata, S., Nelson, G. H., Werner, P. K., Ranken, R. and Harmon, R. C. (
1994
). Anti-Cd31 delays platelet adhesion/aggregation at sites of endothelial injury in mouse cerebral arterioles.
Am. J. Pathol.
145
,
33
-36.
Ruggeri, Z. M. (
1997
). Mechanisms initiating platelet thrombus formation.
Thromb. Haemost.
78
,
611
-616.
Ruggeri, Z. M. (
2002
). Platelets in atherothrombosis.
Nat. Med.
8
,
1227
-1234.
Ryo, R., Yoshida, A., Sugano, W., Yasunaga, M., Nakayama, K., Saigo, K., Adachi, M., Yamaguchi, N. and Okuma, M. (
1992
). Deficiency of P62, a putative collagen receptor, in platelets from a patient with defective collagen-induced platelet aggregation.
Am. J. Hematol.
39
,
25
-31.
Saelman, E. U. M., Kehrel, B., Hese, K. M., Degroot, P. G., Sixma, J. J. and Nieuwenhuis, H. K. (
1994a
). Platelet-adhesion to collagen and endothelial-cell matrix under flow conditions is not dependent on platelet glycoprotein-Iv.
Blood
83
,
3240
-3244.
Saelman, E. U. M., Nieuwenhuis, H. K., Hese, K. M., Degroot, P. G., Heijnen, H. F. G., Sage, E. H., Williams, S., McKeown, L., Gralnick, H. R. and Sixma, J. J. (
1994b
). Platelet-adhesion to collagen type-I through type-Viii under conditions of stasis and flow is mediated by Gpia/Iia (alpha(2)beta(1)-integrin).
Blood
83
,
1244
-1250.
Santoro, S. A. (
1986
). Identification of a 160,000 dalton platelet membrane protein that mediates the initial divalent cation-dependent adhesion of platelets to collagen.
Cell
46
,
913
-920.
Santoro, S. A., Rajpara, S. M., Staatz, W. D. and Woods, V,. Jr (
1988
). Isolation and characterization of a platelet surface collagen binding complex related to VLA-2.
Biochem. Biophys. Res. Commun.
153
,
217
-223.
Santoro, S. A., Walsh, J. J., Staatz, W. D. and Baranski, K. J. (
1991
). Distinct determinants on collagen support alpha 2 beta 1 integrin-mediated platelet adhesion and platelet activation.
Cell Regul.
2
,
905
-913.
Sarkar, S. (
1998
). Tyrosine phosphorylation and translocation of LAT in platelets.
FEBS Lett.
441
,
357
-360.
Savage, B., Saldivar, E. and Ruggeri, Z. M. (
1996
). Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor.
Cell
84
,
289
-297.
Schulte, V., Rabie, T., Prostredna, M., Aktas, B., Gruner, S. and Nieswandt, B. (
2003
). Targeting of the collagen-binding site on glycoprotein VI is not essential for in vivo depletion of the receptor.
Blood
101
,
3948
-3952.
Shattil, S. J., Kashiwagi, H. and Pampori, N. (
1998
). Integrin signaling: the platelet paradigm.
Blood
91
,
2645
-2657.
Shiue, L., Green, J., Green, O. M., Karas, J. L., Morgenstern, J. P., Ram, M. K., Taylor, M. K., Zoller, M. J., Zydowsky, L. D., Bolen, J. B. et al. (
1995
). Interaction of p72syk with the gamma and beta subunits of the high-affinity receptor for immunoglobulin E, Fc epsilon RI.
Mol. Cell. Biol.
15
,
272
-281.
Siljander, P. R. M., Munnix, I. C. A., Smethurst, P. A., Deckmyn, H., Lindhout, T., Ouwehand, W. H., Farndale, R. W. and Heemskerk, J. W. M. (
2004
). Platelet receptor interplay regulates collagen-induced thrombus formation in flowing human blood.
Blood
103
,
1333
-1341.
Sixma, J. J., van-Zanten, G. H., Saelman, E. U., Verkleij, M., Lankhof, H., Nieuwenhuis, H. K. and de-Groot, P. G. (
1995
). Platelet adhesion to collagen.
Thromb. Haemost.
74
,
454
-459.
Sixma, J. J., van-Zanten, G. H., Huizinga, E. G., vanderPlas, R. M., Verkley, M., Wu, Y. P., Gros, P. and deGroot, P. G. (
1997
). Platelet adhesion to collagen: an update.
Thromb. Haemost.
78
,
434
-438.
Smethurst, P. A., Joutsi-Korhonen, L., O'Connor, M. N., Wilson, E., Jennings, N. S., Garner, S. F., Zhang, Y. J., Knight, C. G., Dafforn, T. R., Buckle, A. et al. (
2004
). Identification of the primary collagen-binding surface on human glycoprotein VI by site-directed mutagenesis and by a blocking phage antibody.
Blood
103
,
903
-911.
Snell, D. C., Schulte, V., Jarvis, G. E., Arase, K., Sakurai, D., Saito, T., Watson, S. P. and Nieswandt, B. (
2002
). Differential effects of reduced glycoprotein VI levels on activation of murine platelets by glycoprotein VI ligands.
Biochem. J.
368
,
293
-300.
Staatz, W. D., Rajpara, S. M., Wayner, E. A., Carter, W. G. and Santoro, S. A. (
1989
). The membrane glycoprotein Ia-Iia (Vla-2) complex mediates the Mg++-dependent adhesion of platelets to collagen.
J. Cell Biol.
108
,
1917
-1924.
Stevens, J. M., Jordan, P. A., Sage, T. and Gibbins, J. M. (
2004
). The regulation of integrin-linked kinase in human platelets: evidence for involvement in the regulation of integrin α2β1.
J. Thromb. Haemost.
(in press).
Sugiyama, T., Okuma, M., Ushikubi, F., Sensaki, S., Kanaji, K. and Uchino, H. (
1987
). A novel platelet aggregating factor found in a patient with defective collagen-induced platelet aggregation and autoimmune thrombocytopenia.
Blood
69
,
1712
-1720.
Sullam, P. M., Hyun, W. C., Szollosi, J., Dong, J. F., Foss, W. M. and Lopez, J. A. (
1998
). Physical proximity and functional interplay of the glycoprotein Ib-IX-V complex and the Fc receptor Fc gamma RIIA on the platelet plasma membrane.
J. Biol. Chem.
273
,
5331
-5336.
Sundaresan, P. and Farndale, R. W. (
2003
). Platelet p38 MAP kinase phosphorylation is required by α2β1 through Src family kinases and protein phosphatases.
Platelets
13
,
361
.
Suzuki-Inoue, K., Yatomi, Y., Asazuma, N., Kainoh, M., Tanaka, T., Satoh, K. and Ozaki, Y. (
2001
). Rac, a small guanosine triphosphate-binding protein, and p21-activated kinase are activated during platelet spreading on collagen-coated surfaces: roles of integrin alpha(2)beta(1).
Blood
98
,
3708
-3716.
Suzuki-Inoue, K., Tulasne, D., Shen, Y., Bori-Sanz, T., Inoue, O., Jung, S. M., Moroi, M., Andrews, R. K., Berndt, M. C. and Watson, S. P. (
2002
). Association of Fyn and Lyn with the proline-rich domain of glycoprotein VI regulates intracellular signaling.
J. Biol. Chem.
277
,
21561
-21566.
Tadokoro, S., Shattil, S. J., Eto, K., Tai, V., Liddington, R. C., de Pereda, J. M., Ginsberg, M. H. and Calderwood, D. A. (
2003
). Talin binding to integrin beta tails: a final common step in integrin activation.
Science
302
,
103
-106.
Thai, L. M., Ashman, L. K., Harbour, S. N., Hogarth, P. M. and Jackson, D. E. (
2003
). Physical proximity and functional interplay of PECAM-1 with the Fc receptor Fc gamma RIIa on the platelet plasma membrane.
Blood
102
,
3637
-3645.
Torti, M., Bertoni, A., Canobbio, I., Sinigaglia, F., Lapetina, E. G. and Balduini, C. (
1999
). Rap1B and Rap2B translocation to the cytoskeleton by von Willebrand factor involves Fc gamma II receptor-mediated protein tyrosine phosphorylation.
J. Biol. Chem.
274
,
13690
-13697.
Tsuji, M., Ezumi, Y., Arai, M. and Takayama, H. (
1997
). A novel association of Fc receptor γ-chain with glycoprotein VI and their co-expression as a collagen receptor in human platelets.
J. Biol. Chem.
272
,
23528
-23531.
van de Winkel, J. G. J. and Capel, P. J. A. (
1993
). Human-Ig Fc receptor heterogeneity: molecular aspects and clinical implications.
Immunol. Today
14
,
215
-221.
Wonerow, P., Obergfell, A., Wilde, J. I., Bobe, R., Asazuma, N., Brdicka, T., Leo, A., Schraven, B., Horejsi, V., Shattil, S. J. et al. (
2002
). Differential role of glycolipid-enriched membrane domains in glycoprotein VI- and integrin-mediated phospholipase C gamma 2 regulation in platelets.
Biochem. J.
364
,
755
-765.
Woulfe, D., Jiang, H., Morgans, A., Monks, R., Birnbaum, M. and Brass, L. F. (
2004
). Defects in secretion, aggregation, and thrombus formation in platelets from mice lacking Akt2.
J. Clin. Invest.
113
,
441
-450.
Wu, Y., Suzuki-Inoue, K., Satoh, K., Asazuma, N., Yatomi, Y., Berndt, M. C. and Ozaki, Y. (
2001
). Role of Fc receptor gamma-chain in platelet glycoprotein Ib-mediated signaling.
Blood
97
,
3836
-3845.
Yamaji, S., Suzuki, A., Kanamori, H., Mishima, W., Takabayashi, M., Fujimaki, K., Tomita, N., Fujisawa, S., Ohno, S. and Ishigatsubo, Y. (
2002
). Possible role of ILK-affixin complex in integrin-cytoskeleton linkage during platelet aggregation.
Biochem. Biophys. Res. Commun.
297
,
1324
-1331.
Yamamoto, N., Akamatsu, N., Yamazaki, H. and Tanoue, K. (
1992
). Normal aggregations of glycoprotein-Iv (Cd36)-deficient platelets from seven healthy Japanese donors.
Br. J. Haematol.
81
,
86
-92.
Yap, C. L., Hughan, S. C., Cranmer, S. L., Nesbitt, W. S., Rooney, M. M., Giuliano, S., Kulkarni, S., Dopheide, S. M., Yuan, Y. P., Salem, H. H. et al. (
2000
). Synergistic adhesive interactions and signaling mechanisms operating between platelet glycoprotein Ib/IX and integrin alpha(IIB)beta(3). Studies in human platelets and transfected Chinese hamster ovary cells.
J. Biol. Chem.
275
,
41377
-41388.
Yuan, Y. P., Kulkarni, S., Ulsemer, P., Cranmer, S. L., Yap, C. L., Nesbitt, W. S., Harper, I., Mistry, N., Dopheide, S. M., Hughan, S. C. et al. (
1999
). The von Willebrand factor-glycoprotein Ib/V/IX interaction induces actin polymerization and cytoskeletal reorganization in rolling platelets and glycoprotein Ib/V/IX-transfected cells.
J. Biol. Chem.
274
,
36241
-36251.
Zhang, W., Sloan-Lancaster, J., Kitchen, J., Trible, R. P. and Samelson, L. E. (
1998
). LAT: the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation.
Cell
92
,
83
-92.
Zhao, T. M. and Newman, P. J. (
2001
). Integrin activation by regulated dimerization and oligomerization of platelet endothelial cell adhesion molecule (PECAM)-1 from within the cell.
J. Cell Biol.
152
,
65
-73.