Molecular level studies on platelets deficient in collagen-induced aggregation provide evidence for identifying possible platelet collagen receptors. We investigated platelets from a patient with mild bleeding time prolongation, but otherwise normal coagulation data. Her platelets lacked collagen-induced aggregation and adhesion, but retained normal aggregation and release by other agonists. Labeling her platelets with 125I or 3H and analysis by SDS-PAGE/autoradiography showed normal levels of glycoproteins Ia, Ib, Ila, IIb, IIIa, and IV. However, there were significantly decreased incorporations of both radioactivities into a 61-kD membrane glycoprotein (GP), which was identified as GPVI from its mobility on unreduced-reduced, two-dimensional SDS-PAGE. Sugiyama et al. ( . Blood. 69: 1712 reported that the serum from an idiopathic thrombocytopenic purpura (ITP) patient contained an antibody against a 62-kD platelet protein. Our patient's platelets lacked the antigen for the ITP patient's antibody, demonstrating that the ITP serum contains a specific antibody against GPVI. The patient's parents' platelets contained -50% the normal amount of GPVI, but still had normal collagen-induced aggregation and adhesion. The patient's platelets did not bind to types I and III collagen fibrils. Our results suggest that GPVI functions as a collagen receptor.

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The extracellular matrix protein, laminin, supports platelet adhesion through binding to integrin ␣ 6 ␤ 1 In the present study, we demonstrate that human laminin, purified from placenta, also stimulates formation of filopodia and lamellipodia in human and mouse platelets through a pathway that is dependent on ␣ 6 ␤ 1 and the collagen receptor GPVI. The integrin ␣ 6 ␤ 1 is essential for adhesion to laminin, as demonstrated using an ␣ 6 -blocking antibody, whereas GPVI is dispensable for this response, as shown using "knockout" mouse platelets. On the other hand, lamellipodia formation on laminin is completely inhibited in the absence of GPVI, although filopodia formation remains and is presumably mediated via ␣ 6 ␤ 1 Lamellipodia and filopodia formation are inhibited in Syk-deficient platelets, demonstrating a key role for the kinase in signaling downstream of GPVI and integrin ␣ 6 ␤ 1 GPVI was confirmed as a receptor for laminin using surface plasmon resonance spectroscopy and by demonstration of lamellipodia formation on laminin in the presence of collagenase. These results identify GPVI as a novel receptor for laminin and support a model in which integrin ␣ 6 ␤ 1 brings laminin to GPVI, which in turn mediates lamellipodia formation. We speculate that laminin contributes to platelet spreading in vivo through a direct interaction with GPVI. IntroductionPlatelets play a critical role in the hemostatic process through a combination of adhesion, activation, and aggregation events that lead to formation of a platelet plug and occlusion of the site of damage. Our current understanding of this process highlights a critical role for the interaction of von Willebrand factor (VWF) with the platelet surface receptor, GPIb-IX-V, in mediating platelet tethering at arterial rates of flow. 1,2 This is followed by platelet activation mediated by binding of collagen to the immunoglobulin receptor, GPVI, leading to inside-out stimulation of the integrins, ␣ 2 ␤ 1 and ␣ IIb ␤ 3 . 3 The 2 integrins bind to their respective ligands, collagen and VWF, mediating stable adhesion. Platelet adhesion is further strengthened by integrin-dependent platelet spreading. [4][5][6][7] Platelet aggregation is mediated through binding of fibrinogen to integrin ␣ IIb ␤ 3 in a process dependent on release of the secondary mediators, ADP and thromboxanes. [8][9][10] The contribution of other extracellular matrix proteins to the hemostatic process is unclear. Recently, however, Nieswandt's group 11 has highlighted a possible role for the 2 ␤ 1 -integrins, ␣ 5 ␤ 1 and ␣ 6 ␤ 1 , which are receptors for fibronectin and laminin, respectively, in supporting adhesion in vivo in concert with integrins ␣ 2 ␤ 1 and ␣ IIb ␤ 3 . This was achieved by comparing platelet adhesion and aggregate formation in vivo using fluorescent intravital microscopy in mice deficient in ␣ 2 and ␤ 1 integrin subunits and in the presence of ␣ IIb ␤ 3 blockade. 11 This study therefore illustrates that laminin, fibronectin, and other ligands for ␣ 5 ␤ 1 and ␣ 6 ␤ 1 may contribute to...

Background: Platelet collagen receptor GPVI likely functions as a dimer rather than a monomer. Results: Preformed GPVI dimers, but not monomers, in resting platelets bind specific collagen sequences and are essential for platelet adhesion and activation. Conclusion: Constitutive GPVI dimers on resting platelets support platelet adhesion to collagen and activation. Significance: Resting platelets bind collagen through GPVI dimers, allowing immediate initiation of thrombus formation.

The glycoprotein VI (GPVI)-Fc receptor (FcR)␥The adhesion and activation of platelets by subendothelial collagen fibers initiates aggregate formation at sites of vessel damage. Glycoprotein (GP) 1 VI plays a critical role in the activatory events induced by collagen as shown by the lack of response to collagen in human and mice platelets deficient in the glycoprotein (1, 2). A collagen-related peptide and a snake venom toxin, convulxin, interact specifically with GPVI and mimic many of the responses to collagen (3-5).Because of the physiological importance of GPVI, the mechanism of the GPVI-mediated signaling system has been extensively investigated (6 -8). GPVI is present as a complex with Fc receptor (FcR) ␥-chain in the platelet membrane (8 -10). The Src family kinases, Fyn and Lyn, are associated with GPVIFcR ␥-chain complex in platelets and initiate activation through phosphorylation of the immunoreceptor tyrosinebased activation motif (ITAM) in the FcR ␥-chain leading to binding and activation of the tyrosine kinase Syk. A series of adapter molecules including LAT and SLP76 orchestrate a carefully regulated signaling network leading to activation of PLC␥2, phosphoinositol 3-kinase, and small molecular weight G proteins, leading to platelet activation (6, 7).The cloning of GPVI (11-14) has revealed it to be a member of the immunoglobulin (Ig) superfamily, showing close homology to Fc␣RI. GPVI has a charged arginine residue in its transmembrane domain. This arginine, together with elements within the cytoplasmic domain, is crucial for association of GPVI with FcR ␥-chain and GPVI-mediated signal transduction (15,16). In addition, the cytoplasmic tail of GPVI has a cluster of 6 proline residues of unknown function (11)(12)(13)(14). This sequence of GPVI, RPLPPLPPLP, contains a consensus Src family kinase-SH3 recognition motif (RPLPPLP) (17,18), and provides a potential site of interaction with Fyn and Lyn via their SH3 domains.In this study, we demonstrate that depletion of the prolinerich domain in GPVI abolishes the association with Fyn and Lyn and prevents tyrosine phosphorylation of FcR ␥-chain and downstream responses. From these findings, we suggest that Fyn/Lyn directly bind the proline-rich domain of GPVI and that this association is necessary for phosphorylation of the ITAM and downstream signals.

Evidence was obtained about the mechanism responsible for platelet integrin ␣ 2 ␤ activation by determining effects of various inhibitors on soluble collagen binding, a parameter to assess integrin ␣ 2 ␤ 1 activation, in stimulated platelets. Agonists that can also activate platelet glycoprotein IIb/IIIa are able to activate integrin ␣ 2 ␤ 1 , but those operating via glycoprotein Ib cannot. Activation of ␣ 2 ␤ 1 induced by low thrombin or collagen-related peptide concentrations was almost completely inhibited by apyrase, and the inhibitors wortmannin, 4-amino-5-(chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, bisindolylmaleimide I, and SQ29548 significantly inhibited it. Activation induced by high thrombin or collagen-related peptide concentrations was far less sensitive to these inhibitors. However, only wortmannin markedly inhibited ADP-induced integrin ␣ 2 ␤ 1 activation, and this was not ADP concentration-dependent. These results suggest that at the low agonist concentrations, the released ADP would be a primary inducer of integrin ␣ 2 ␤ 1 activation, while at the high agonist concentrations, there would be several pathways through which integrin ␣ 2 ␤ 1 activation can be induced. Kinetic analyses revealed that ADP-induced platelets had about the same number of binding sites (B max ) as thrombin-induced platelets, but their affinity (K d ) for soluble collagen was 3.7-12.7-fold lower, suggesting that activated integrin ␣ 2 ␤ 1 induced by ADP is different from that induced by thrombin. The data are consistent with an activation mechanism involving released ADP and in which there exists two different states of activated integrin ␣ 2 ␤ 1 ; these activated forms of integrin ␣ 2 ␤ 1 would have different conformations that determine their ligand affinity.Integrins comprise a family of heterodimeric cell surface proteins that mediate intracellular and cell-to-extracellular interactions. In humans, at least 15 different ␣-subunits and eight different ␤-subunits have been identified to date. The various permutations of the ␣-and ␤-subunit complexes yield integrin dimers with diverse ligand specificities and biological activities. There is tissue-specific expression of each type of integrin; some integrins are only expressed in a certain tissue, while others are more universal.Integrin ␣ IIb ␤ 3 (platelet glycoprotein (GP) 1 IIb/IIIa) is only expressed in platelets and megakaryocytes, but integrin ␣ 2 ␤ 1 (platelet GP Ia/IIa) is known to be present in many cell types (1). The GP IIb/IIIa complex is present as a nonactive heterodimer in resting platelets and becomes activated when platelets are induced by agonists (2-4); activated GP IIb/IIIa possesses high affinity for its ligand, fibrinogen. GP IIb/IIIa is one of the most abundant proteins in the platelet membrane, and its binding reaction with fibrinogen was shown to be one of the most important reactions in platelet aggregation. On the other hand, although integrin ␣ 2 ␤ 1 was indicated to be a receptor for collagen from studies on a patient's platelets lacking...

Platelet interaction with soluble and insoluble collagens was characterized through binding studies. In contrast to resting platelets, cells reacted with activators, TS2/16 (integrin ␣ 2 ␤ 1 -activating antibody), thrombin, collagen-related peptide, or ADP, exhibited specific soluble collagen binding that is Mg 2؉ -dependent, but inhibited by prostaglandin I 2 , Ca 2؉ , and Gi9 (anti-integrin ␣ 2 ␤ 1 antibody). Each platelet has 1500 -3500 soluble collagen binding sites, with a dissociation constant of 3.5-9 ؋ 10 ؊8 M. This is the first study to show the specific binding of soluble collagen to platelets; our data strongly suggest that the receptor is integrin ␣ 2 ␤ 1 after it becomes activated upon platelet activation. These results suggest that activation of platelets transforms integrin ␣ 2 ␤ 1 to a state with higher affinity binding sites for soluble collagen. The soluble collagen-platelet interaction was compared with the platelet interaction with fibrillar collagen, which has until now not been demonstrated to bind specifically to platelets. Here, we demonstrated specific, biphasic fibrillar collagen binding. One phase is rapid and metal ion-independent, and accounts for most of the binding. The other phase is slow and Mg 2؉ -dependent. The characteristic differences in the specific bindings of soluble and fibrous collagens demonstrate the different contributions of two different collagen receptors.When platelets react with collagen, they form aggregates after activation of their signal transduction systems through processes involving many reactions including calcium release, formation of phosphoinositides, and phosphorylation of proteins. Platelets can also adhere to an immobilized collagen surface. Collagen is one of the main components of the vascular subendothelium and becomes exposed to the blood stream when endothelial cells are damaged. These observations suggest that the interaction of platelets with collagen is one of the first, requisite reactions in thrombus formation under physiological conditions. The interaction of platelets with collagen has been studied under two conditions. One approach is to analyze platelet adhesion and aggregation onto a collagen-coated surface under flow conditions using whole blood (1-5). This method observes platelet adhesion under flow conditions that closely simulate physiological conditions, and the fact that platelets adhere and aggregate under these conditions shows that the interaction with collagen is involved in physiological thrombus formation. Alternatively, platelet adhesion on a collagen-coated surface can be analyzed under static conditions; this type of assay uses washed platelets instead of whole blood (6, 7). In both systems, two platelet glycoproteins, glycoprotein (GP) 1 Ia/IIa, also known as integrin ␣ 2 ␤ 1 , and GP VI were found to participate in platelet adhesion to the collagen surface.However, the direct binding of collagen to platelets has not been studied in detail because soluble collagen did not bind to platelets. Resting platelets do not...

Platelet glycoprotein VI (GPVI), a 62kD membrane protein, has been identified as one of the platelet receptors for collagen, since GPVI-deficient platelets exhibit abnormal responses to collagen and an abnormal bleeding tendency. We report a female patient with a mild bleeding history whose platelets expressed 10% GPVI of normal platelets. Shape change, aggregation and ATP release of the patient's platelets were completely absent in response to 1-5 micrograms/ml collagen but present normally in response to ADP and Ca2+ ionophore A23187. Adhesion of the patient's platelets to coated collagen was mildly affected (40-60% of normal platelets) in spite of only 10% expression of GPVI. Flow cytometrical studies revealed that the patient's platelets expressed normal amounts of the GPIa/IIa complex. These results suggest that platelet GPVI is less involved in adhesion to collagen than shape change and aggregation induced by collagen.