Identification of P2Y12-dependent and -independent mechanisms of glycoprotein VI–mediated Rap1 activation in platelets

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Abstract
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 αIIbβ3 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 FcRγ but independent of another platelet collagen receptor, α2β1. Interestingly, GPVI-mediated Rap1 activation in human platelets is largely dependent on adenosine diphosphate (ADP) signaling through the P2Y12 and not the P2Y1 receptor. However, experiments with specific ADP receptor antagonists and platelets from knockout mice deficient in P2Y1 or the P2Y12-associated G-protein, Gαi2, indicate that human and murine platelets also have a significant P2Y12-independent component of GPVI-mediated Rap1 activation. The P2Y12-independent component is dependent on phosphatidylinositol 3-kinase and is augmented by epinephrine-mediated signaling. P2Y12-dependent and -independent components are also observed in GPVI-mediated platelet aggregation, further supporting a role for Rap1 in aggregation. These results define mechanisms of GPVI-mediated platelet activation and implicate Rap1 as a key signaling protein in GPVI-induced platelet signaling.