Platelet activation is characterized by shape change, induction of fibrinogen receptor expression and release of granular contents, leading to aggregation and plug formation. While this response is essential for hemostasis, it is also important in the pathogenesis of a broad spectrum of diseases, including myocardial infarction, stroke and unstable angina. Adenosine 5'-diphosphate (ADP) induces platelet aggregation, but the mechanism for this has not been established, and the relative contribution of ADP in hemostasis and the development of arterial thrombosis is poorly understood. We show here that the purinoceptor P2Y1 is required for platelet shape change in response to ADP and is also a principal receptor mediating ADP-induced platelet aggregation. Activation of P2Y1 resulted in increased intracellular calcium but no alteration in cyclic adenosine monophosphate (cAMP) levels. P2Y1-deficient platelets partially aggregated at higher ADP concentrations, and the lack of P2Y1 did not alter the ability of ADP to inhibit cAMP, indicating that platelets express at least one additional ADP receptor. In vivo, the lack of P2Y1 expression increased bleeding time and protected from collagen- and ADP-induced thromboembolism. These findings support the hypothesis that the ATP receptor P2Y1 is a principal receptor mediating both physiologic and pathological ADP-induced processes in platelets.

No abstract

Both iPF(2alpha)-III and iPE(2)-III exert their effects on platelet function and vascular tone in vivo by acting as incidental ligands at membrane TPs rather than via a distinct iP receptor. Activation of TPs by iPs may be of importance in syndromes in which cyclooxygenase activation and oxidant stress coincide, such as in atherosclerosis and reperfusion after tissue ischemia.

Glycoprotein (GP) VI is a critical platelet collagen receptor, yet the steps involved in GPVI-mediated platelet activation remain incompletely understood. Because activation of Rap1, an abundant small guanosine triphosphatase (GTPase) in platelets, contributes to integrin ␣ IIb ␤ 3 activation, we asked whether and how GPVI signaling activates Rap1 in platelets. Here we show that platelet Rap1 is robustly activated upon addition of convulxin, a GPVI-specific agonist. Using a reconstituted system in RBL-2H3 cells, we found that GPVI-mediated Rap1 activation is dependent on FcR␥ but independent of another platelet collagen receptor, ␣ 2 ␤ 1 . Interestingly, GPVI-mediated Rap1 activation in human platelets is largely dependent on adenosine diphosphate (ADP) signaling through the P2Y 12 and not the P2Y 1 receptor. However, experiments with specific ADP receptor antagonists and platelets from knockout mice deficient in P2Y 1 or the P2Y 12 -associated G-protein, G␣i 2 , indicate that human and murine platelets also have a significant P2Y 12 -independent component of GPVImediated Rap1 activation. The P2Y 12 -independent component is dependent on phosphatidylinositol 3-kinase and is augmented by epinephrine-mediated signaling. P2Y 12 -dependent and -independent components are also observed in GPVImediated platelet aggregation, further supporting a role for Rap1 in aggregation. These results define mechanisms of GPVImediated platelet activation and implicate Rap1 as a key signaling protein in GPVI IntroductionCollagen is a major component of the subendothelial matrix and atherosclerotic plaques and is a potent platelet agonist that contributes to thrombus formation upon plaque rupture. Collagenmediated platelet activation results from a complex set of signals initiated by the coordination of platelet surface proteins, including the immunoglobulin superfamily receptor glycoprotein (GP) VI, the integrin ␣ 2 ␤ 1 , and most recently, GPV. [1][2][3] Activation of GPVI causes the assembly of distinct proximal signaling pathways. GPVI is constitutively associated with FcR␥, a protein containing an immunoreceptor tyrosine-based activation motif. 4,5 Upon collagen ligation to GPVI, FcR␥ becomes phosphorylated by a Src family kinase, 6,7 which subsequently allows the recruitment of other signaling proteins such as Syk, Bruton tyrosine kinase, and phospholipase C␥. [6][7][8][9][10][11] GPVI-mediated signaling is absolutely required for collageninduced platelet aggregation. Human platelets lacking GPVI, but expressing ␣ 2 ␤ 1 , fail to aggregate in response to collagen. 12 In addition, GPVI antagonists inhibit collagen-mediated aggregation. 13 Although human platelets lacking ␣ 2 ␤ 1 also fail to aggregate in response to collagen, 14 fibrillar collagen-mediated platelet aggregation still occurs in ␤ 1 -deficient murine platelets that express GPVI. 15,16 Additionally, GPVI signaling itself can activate ␣ 2 ␤ 1 function. 15 This finding suggests that not only is GPVI a key signaling component of collagen-mediated platelet activation, ...