Summary. Background: Platelet ADP receptor P2Y 12 is well studied and recognized as a key player in platelet activation, hemostasis and thrombosis. However, the role of P2Y 12 in platelet apoptosis remains unknown. Objectives: To evaluate the role of the P2Y 12 receptor in platelet apoptosis. Methods: We used flow cytometry and Western blotting to assess apoptotic events in platelets treated with ABT-737 or ABT-263, and stored at 37°C, combined with P2Y 12 receptor antagonists or P2Y 12 -deficient mice. Results: P2Y 12 activation attenuated apoptosis induced by ABT-737 in human and mouse platelets in vitro, evidenced by reduced phosphatidylserine (PS) exposure, diminished depolarization of mitochondrial inner transmembrane potential (DYm) and decreased caspase-3 activation. Through increasing the phosphorylation level of Akt and Bad, and changing the interaction between different Bcl-2 family proteins, P2Y 12 activation inactivated Bak/Bax. This antiapoptotic effect could be abolished by P2Y 12 antagonism or PI3K inhibition. We also observed the antiapoptotic effect of P2Y 12 activation in platelets stored at 37°C. P2Y 12 activation improved the impaired activation responses of apoptotic platelets stressed by ABT-737. In platelets from mice dosed with ABT-263 in vivo, clopidogrel or deficiency of P2Y 12 receptor enhanced apoptosis along with increased Bak/Bax activation. Conclusions: This study demonstrates that P2Y 12 activation protects platelets from apoptosis via PI3k-dependent Bak/Bax inactivation, which may be physiologically important to counter the proapoptotic challenge. Our findings that P2Y 12 blockade exaggerates platelet apoptosis induced by ABT-263 (Navitoclax) also imply a novel drug interaction of antagonists.

Summary Background In our previous in vitro study we reported a constitutively active chimeric P2Y12 receptor (cP2Y12) and found AR-C78511 is a potent inverse agonist at this receptor. The role of this cP2Y12 receptor in platelet activation and thrombosis is not clear. Objectives To investigate the physiological implications of the constitutively active P2Y12 receptor in platelet activation, thrombus formulation and evaluate the antiplatelet activity of AR-C78511 as an inverse agonist. Methods and Results We generated transgenic mice conditionally and platelet-specifically expressing cP2Y12. High expression of cP2Y12 receptor in platelets increased platelet reactivity as evidenced by increased platelet aggregation in response to multiple platelet agonists. Moreover, transgenic mice displayed shortened bleeding time, more rapid and stable thrombus formation in mesenteric artery injured with FeCl3. The constitutive activity of cP2Y12 in platelets was confirmed by decreased platelet cAMP levels and constitutive Akt phosphorylation in the absence of agonists. AR-C78511 reversed the cAMP decrease in transgenic mouse platelets, and exhibited superior antiplatelet effect over AR-C69931MX in transgenic mice. Conclusions These findings further emphasize the importance of P2Y12 in platelet activation, hemostasis and thrombosis, as well as the prothrombotic role of constitutive activity of P2Y12. Our data also validates the in vivo inverse agonist activity of AR-C78511 and confirms its superior antiplatelet activity over neutral antagonist.