Premature Platelet Activation and Resistance to P2Y12 Inhibitors in Rasa3 Mutant Mice

The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the guanine nucleotide exchange factor CalDAG-GEFI and an unknown regulator operating downstream of the ADP receptor, P2Y12, the target of antithrombotic therapy. Here we provide evidence that the GTPase-activating protein, RASA3, is a critical inhibitor of platelet activation and the missing link in the P2Y12/RAP1 signaling pathway. Genetic inactivation of Rasa3 led to premature activation and markedly reduced lifespan of circulating platelets in mice (t_1/2=14 hrs vs. 55 hrs in controls). The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Importantly, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Thus, constitutively active RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling. At sites of vascular injury, P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation. Our findings provide critical mechanistic insights for the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders.