Both Platelet ADP Receptors Contribute to Microparticle Formation.

Abstract

Microparticles are shed from the platelet membrane upon platelet activation, and they contain negatively charged phospholipids such as phosphatidylserine (PS) on their membrane that bind to coagulation factors and aid in clot formation. While the procoagulant nature of microparticles is clear, the relative contributions of platelet agonists to microparticle formation leading to a thrombus are still unclear. Because secreted ADP from the dense granules has been shown to potentiate granule release, GPIIb/IIIa activation, and platelet thrombin generation, we studied the effect of antagonizing the P2Y1 and P2Y12 receptors on microparticle formation from platelets that are stimulated with convulxin and thrombin. It has been previously shown that a deficiency in the P2Y12 receptor causes an increase in bleeding time, and we found prolonged bleeding times with P2Y1 knockout mice when compared to the wild type mice. Because microparticles are a part of the hemostatic process, we set out to see if there was a contribution of the P2Y receptors on agonist-induced platelet microparticle formation. Flow cytometry was used to measure microparticle formation in human blood in the presence or absence of P2Y1 or P2Y12 receptor antagonists. In addition, we measured agonist-induced microparticle formation in wild type mouse blood and compared the microparticle formation to P2Y1 knockout mouse blood microparticle. Using human blood stimulated with thrombin and convulxin we observed a decrease in microparticle count when administering the P2Y1 antagonist MRS2179 by 17%, and the P2Y12 antagonist AR-C69931MX by 28%. Furthermore, there was a decrease in microparticle formation in P2Y1 knockout mouse blood when compared to the wild type mouse microparticle formation by 13%. These results illustrate that both of the P2Y1 and P2Y12 receptors play an intricate role in microparticle formation in vivo, and may be one more effect underlying the extended bleeding times of patients deficient in the P2Y12 receptor or for the slightly increased bleeding of those taking the P2Y12 receptor antagonist clopidogrel.