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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.

The human P2Y! purinoceptor has been expressed in Jurkat cells and the effects of HPLC purified nucleotides on calcium movements were measured. The most potent agonist was 2-methylthio-ADP followed by ADP. ATP, Sp-ATPctS and ß,^ methylene-ATP were competitive antagonists. Suramin and PPADS inhibited the effects of ADP. This pharmacological profile is the same as that of the so-called P2T purinoceptor responsible for platelet aggregation, which has not yet been identified. Using PCR we found the P2Y] receptor to be present in blood platelets and megakaryoblastic cell lines. These data suggest that the P2Yx receptor may be the elusive P2T receptor.

See also Brill A. A ride with ferric chloride. This issue, pp 776–8. Summary.  Background: The FeCl3‐induced vascular injury model is widely used to study thrombogenesis in vivo, but the processes leading to vascular injury and thrombosis are poorly defined. Objectives: The aim of our study was to better characterize the mechanisms of FeCl3‐induced vascular injury and thrombus formation, in order to evaluate the pathophysiological relevance of this model. Methods: FeCl3 was applied at different concentrations (from 7.5% to 20%) and for different time periods (up to 5 min) to mouse carotid or mesenteric arteries. Results: Under all the conditions tested, ultrastructural analysis revealed that FeCl3 diffused through the vessel wall, resulting in endothelial cell denudation without exposure of the inner layers. Hence, only the basement membrane components were exposed to circulating blood cells and might have contributed to thrombus formation. Shortly after FeCl3 application, numerous ferric ion‐filled spherical bodies appeared on the endothelial cells. Interestingly, platelets could adhere to these spheres and form aggregates. Immunogold labeling revealed important amounts of tissue factor at their surface, suggesting that these spheres may play a role in thrombin generation. Invitro experiments indicated that FeCl3 altered the ability of adhesive proteins, including collagen, fibrinogen and von Willebrand factor, to support platelet adhesion. Finally, real‐time intravital microscopy showed no protection against thrombosis in GPVI‐immunodepleted and β1−/− mice, suggesting that GPVI and β1 integrins, known to be involved in initial platelet adhesion and activation, do not play a critical role in FeCl3‐induced thrombus formation. Conclusion: This model should be used cautiously, in particular to study the earliest stage of thrombus formation.

Mutations in the MYH9 gene encoding the nonmuscle myosin heavy chain IIA result in bleeding disorders characterized by a macrothrombocytopenia. To understand the role of myosin in normal platelet functions and in pathology, we generated mice with disruption of MYH9 in megakaryocytes. MYH9⌬ mice displayed macrothrombocytopenia with a strong increase in bleeding time and absence of clot retraction. However, platelet aggregation and secretion in response to any agonist were near normal despite absence of initial platelet contraction. By contrast, integrin outside-in signaling was impaired, as observed by a decrease in integrin ␤3 phosphorylation and PtdIns(3,4)P 2 accumulation following stimulation. Upon adhesion on a fibrinogen-coated surface, MYH9⌬ platelets were still able to extend lamellipodia but without stress fiber-like formation. As a consequence, thrombus growth and organization, investigated under flow by perfusing whole blood over collagen, were strongly impaired. Thrombus stability was also decreased in vivo in a model of FeCl 3 -induced injury of carotid arteries. Overall, these results demonstrate that while myosin seems dispensable for aggregation and secretion in suspension, it plays a key role in platelet contractile phenomena and outsidein signaling. These roles of myosin in platelet functions, in addition to thrombocytopenia, account for the strong hemostatic defects observed in MYH9⌬ mice. IntroductionImportant morphologic changes occur in platelets during their activation at sites of vascular injury. The cells lose their resting discoid shape to become spheroid and contracted, emitting membrane blebs and longer extensions. [1][2][3][4] Once in contact with a surface, the spheroid platelets extend long filopodia and finally spread over it by emitting thin, sheet-like lamellipodia. 1,2 Myosin activation plays a central role in the cytoskeletal rearrangements underlying these changes in morphology. Myosin becomes activated after phosphorylation of the myosin regulatory light chain (RLC), which results from both calcium-regulated myosin lightchain kinase activity and Rho kinase-regulated myosin phosphatase activity. [5][6][7][8] Activated myosin assembles into short filaments through the myosin heavy chain and interacts mainly with central actin filaments. Myosin has been proposed to participate in several platelet contractile functions such as platelet spheration, contraction and stress-fiber formation, and fibrin clot retraction. Platelet spheration and contraction, as observed in the aggregometer, closely correlate with phosphorylation of the RLC 9,10 and are prevented when RLC phosphorylation is inhibited. 6,7,9,10 Myosin has also been shown to be associated with stress fiber-like structures in spreading adherent platelets. 11 In addition, myosin could play a role in platelet secretion, as it is decreased by inhibition of myosin RLC phosphorylation. 5,[12][13][14][15] Finally, a role of myosin in clot retraction has long been suspected in view of the necessity for a contractile force and was ...

The human P2Y1 receptor heterologously expressed in Jurkat cells behaves as a specific adenosine 5′-diphosphate (ADP) receptor at which purified adenosine triphosphate (ATP) is an ineffective agonist, but competitively antagonizes the action of ADP. This receptor is thus a good candidate to be the elusive platelet P2T receptor for ADP. In the present work, we examined the effects on ADP-induced platelet responses of two selective and competitive P2Y1 antagonists, adenosine-2′-phosphate-5′-phosphate (A2P5P) and adenosine-3′-phosphate-5′-phosphate (A3P5P). Results were compared with those for the native P2Y1 receptor expressed on the B10 clone of rat brain capillary endothelial cells (BCEC) and for the cloned human P2Y1 receptor expressed on Jurkat cells. A2P5P and A3P5P inhibited ADP-induced platelet shape change and aggregation (pA2 = 5) and competitively antagonized calcium movements in response to ADP in fura-2–loaded platelets, B10 cells, and P2Y1-Jurkat cells. In contrast, these compounds had no effect on ADP-induced inhibition of adenylyl cyclase in platelets or B10 cells, whereas known antagonists of platelet activation by ADP such as Sp-ATPαS were effective. These identical signaling responses and pharmacologic properties suggest that platelets and BCEC share a common P2Y1 receptor involved in ADP-induced aggregation and vasodilation, respectively. This P2Y1 receptor coupled to the mobilization of intracellular calcium stores was found to be necessary to trigger ADP-induced platelet aggregation. The present results, together with data from the literature, also point to the existence of another as yet unidentified ADP receptor, coupled to adenylyl cyclase and responsible for completion of the aggregation response. Thus, the term, P2T, should no longer be used to designate a specific molecular entity.

Pharmacological properties of the human P2Y1 receptor transfected in Jurkat cells and of the endogenous receptor in rat brain capillary endothelial cells were analyzed under conditions in which the purity of adenine triphosphate nucleotides was controlled by creatine phosphate/creatine phosphokinase (CP/CPK). ATP, a partial agonist of the receptor, was inactive in the presence of CP/CPK. Results further indicated that ATP was a competitive antagonist of ADP actions. Ki values were 23.0 +/- 1.5 microM in endothelial cells and 14.3 +/- 0.3 microM in Jurkat cells. Solutions prepared from commercially available 2-methylthio-ATP (2-MeSATP) or 2-chloro-ATP (2-ClATP) contained approximately 10% of ADP derivatives. ADP derivatives were removed from the solution by treatment with CP/CPK. Purified 2-MeSATP and 2-ClATP antagonized platelet aggregation induced by ADP. They did not activate P2Y1 receptors but prevented ADP actions in a competitive manner. Ki values for 2-MeSATP were 36. 5 microM in endothelial cells and 5.7 +/- 0.4 microM in Jurkat cells, and Ki values for 2-ClATP were 27.5 microM in endothelial cells and 2.3 +/- 0.3 microM in Jurkat cells. EDTA potentiated actions of ADP and ATP on endothelial cells by 2.4- and 3.6-fold, respectively. In conclusion, the rat and human P2Y1 receptors are ADP-specific receptors that recognize ADP and 2-methylthio-ADP, whereas ATP, 2-MeSATP, and 2-ClATP are competitive antagonists. The results further point to the close pharmacological similarity of the P2Y1 receptor and the platelet ADP receptor.

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