The P2X1 receptor is a fast ATP-gated cation channel expressed in blood platelets, where its role has been difficult to assess due to its rapid desensitization and the lack of pharmacological tools. In this paper, we have used P2X1 −/− and wild-type mouse platelets, treated with apyrase to prevent desensitization, to demonstrate the function of P2X1 in the response to thrombogenic stimuli. In vitro, the collagen-induced aggregation and secretion of P2X1-deficient platelets was decreased, as was adhesion and thrombus growth on a collagen-coated surface, particularly when the wall shear rate was elevated. In vivo, the functional role of P2X1 could be demonstrated using two models of platelet-dependent thrombotic occlusion of small arteries, in which blood flow is characterized by a high shear rate. The mortality of P2X1 −/− mice in a model of systemic thromboembolism was reduced and the size of mural thrombi formed after a laser-induced vessel wall injury was decreased as compared with normal mice, whereas the time for complete thrombus removal was shortened. Overall, the P2X1 receptor appears to contribute to the formation of platelet thrombi, particularly in arteries in which shear forces are high.

Glycoprotein V (GPV) is a subunit of the platelet GPIb-V-IX receptor for von Willebrand factor and thrombin. GPV is cleaved from the platelet surface during activation by thrombin, but its role in hemostasis is still unknown. It is reported that GPV knockout mice had a decreased tendency to form arterial occluding thrombi in an intravital thrombosis model and abnormal platelet interaction with the subendothelium. In vitro, GPV-deficient platelets exhibited defective adhesion to a collagen type I-coated surface under flow or static conditions. Aggregation studies demonstrated a decreased response of the GPV-deficient platelets to collagen, reflected by an increased lag phase and reduced amplitude of aggregation. Responses to adenosine diphosphate, arachidonic acid, and the thromboxane analog U46619 were normal but were enhanced to low thrombin concentrations. The defect of GPV null platelets made them more sensitive to inhibition by the anti-GPVI monoclonal antibody (mAb) JAQ1, and this was also the case in aspirin-or apyrase-treated platelets. Moreover, an mAb (V.3) against the extracellular domain of human GPV selectively inhibited collagen-induced aggregation in human or rat platelets. V.3 injected in rats as a bolus decreased the ex vivo collagen aggregation response without affecting the platelet count. Finally, surface plasmon resonance studies demonstrated binding of recombinant soluble GPV on a collagen-coupled matrix. In conclusion, GPV binds to collagen and appears to be required for normal platelet responses to this agonist. IntroductionPlatelets play a central role in hemostasis through their ability to adhere to a damaged vessel wall and to aggregate in response to agonists such as thrombin, collagen, or adenosine diphosphate (ADP), 1 and these properties are known to be mediated by cell surface glycoproteins. 2,3 However, the exact functions of numerous platelet glycoproteins that have been characterized biochemically remain unknown. Glycoprotein V (GPV, Mr 82 kd) is one of the most abundant glycoproteins at the surface of blood platelets and has long been identified, 4-6 but its functional role is still subject to speculation. GPV is noncovalently linked to the GPIb-IX von Willebrand factor (vWF) receptor on the platelet surface. 7,8 This type I transmembrane protein has a large extracellular domain comprising 15 Leu-rich motifs followed by a thrombin cleavage site. 9,10 The specific release of a soluble 69-kd extracellular domain fragment (GPVf1) by thrombin has led to its proposal as a thrombin receptor, 6 whereas cloning of the gene in rat and mouse revealed a well-conserved thrombin cleavage site. 11 Studies in transfected cells have shown that GPV is required for efficient thrombin binding, possibly through a direct interaction with GPIb␣. 12,13 The release of a soluble fragment by thrombin has been used to develop a specific enzyme-linked immunosorbent assay for GPV that is being tested as a means of monitoring platelet activation under conditions of clinical thrombosis. 14 The role of GPV a...