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...

Objective— Platelet activation occurs in response to adhesion receptors for von Willebrand factor (GPIb-V-IX) and collagen (GPVI and α 2 β 1 integrin) acting upstream of phospholipase C (PLC) γ2. However, PLCβ transduces signals from Gαq protein-coupled receptors for soluble agonists (P2y 1 , TxA 2 /TP, and thrombin/PAR). A Gi-dependent pathway amplifies most of these responses. Methods and Results— To evaluate the role of adhesion receptors signaling in arterial thrombosis, PLCγ2 knockout mice were studied in blood perfusion assays over fibrillar collagen and in a laser-induced mesenteric artery model of thrombosis. In vitro, PLCγ2-deficient platelets formed a single layer incapable of generating a thrombus on collagen, whereas Gαq-deficient platelets formed reduced size aggregates compared with wild-type cells. In the in vivo model, PLCγ2 −/− mice displayed defective thrombus formation in superficial lesions but productive thrombosis after a more severe laser injury. In contrast, resistance to thrombosis was observed in Gαq −/− mice in both levels of injury. Conclusions— These results demonstrate that signaling through PLCγ2 plays an important role in arterial thrombosis, but that its contribution depends on the severity of the vascular lesion.

Summary.  Previous studies in experimental models revealed a role for the P2Y1 platelet ADP receptor in systemic vascular thromboembolism models. In the present work, we used models of localized arterial and venous thrombosis to assess the role of the P2Y1 receptor in these processes. Arterial thrombosis was induced in one mesenteric arteriole of a mouse using FeCl3, while venous thrombosis was studied in a Wessler model adapted to rats. P2Y1‐deficient mice and mice treated with the P2Y1 antagonist MRS2179 displayed significantly less arterial thrombosis than their respective controls. Combination of P2Y1 deficiency with P2Y12 inhibition led to a significant additive effect. Venous thrombosis was slightly but significantly inhibited in MRS2179‐treated rats. These results demonstrate a role for the P2Y1 receptor in both arterial and venous thrombosis, further establishing this receptor as a potential target for antithrombotic drugs.

Platelets are critical for normal hemostasis. Their deregulation can lead to bleeding or to arterial thrombosis, a primary cause of heart attack and ischemic stroke. Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) is a 5-phosphatase capable of dephosphorylating the phosphatidylinositol 3,4,5-trisphosphate second messenger into phosphatidylinositol 3,4-bisphosphate. SHIP1 plays a critical role in regulating the level of these 2 lipids in platelets. Using SHIP1-deficient mice, we found that its loss affects platelet aggregation in response to several agonists with minor effects on fibrinogen binding and β 3 integrin tyrosine phosphorylation. Accordingly, SHIP1-null mice showed defects in arterial thrombus formation in response to a localized laser-induced injury. Moreover, these mice had a prolonged tail bleeding time. Upon stimulation, SHIP1-deficient platelets showed large membrane extensions, abnormalities in the open canalicular system, and a dramatic decrease in close cell-cell contacts. Interestingly, SHIP1 appeared to be required for platelet contractility, thrombus organization, and fibrin clot retraction. These data indicate that SHIP1 is an important element of the platelet signaling machinery to support normal hemostasis. To our knowledge, this is the first report unraveling an important function of SHIP1 in the activation of hematopoietic cells, in contrast to its well-documented role in the negative regulation of lymphocytes. IntroductionPlatelet activation is a highly regulated process and critical for mediating normal hemostasis. Vascular injury exposes subendothelial matrix proteins that allow platelet arrest and their subsequent spreading, activation, and secretion of soluble mediators. These soluble agonists recruit circulating platelets, allowing aggregation for the bleeding to stop. In pathological states, platelet activation can contribute to thrombosis and cerebrovascular and arterial occlusion and eventually to heart attacks and strokes. Several signaling mechanisms have been shown to contribute to platelet activation, including the phospholipase C (PLC)/Ca 2+ and the PI3K pathways. By generating phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] and phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P 2 ] second messengers, PI3K plays an important role in platelet activation and plug stability (1-3). Using a selective PI3K β inhibitor, Jackson et al. (3) recently suggested that this lipid kinase could be a potential target for antithrombotic therapy.It is now well established in several models that phosphoinositide phosphatases involved in the hydrolysis of PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 , such as the phosphatase and tensin homolog (PTEN), are essential for the negative regulation of PI3K pathways (4). PTEN is commonly deleted or inactivated in several cancers,

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