Collagen-induced generation of platelet-derived microparticles in whole blood is dependent on ADP released from red blood cells and calcium ions

We have evaluated the effects of different anti-coagulants or agonists on the generation of platelet-derived microparticles (PMPs) using flow cytometry. Twenty mug/ml of collagen induced significantly greater PMP formation in whole blood anti-coagulated with argatroban, a selective thrombin inhibitor, as compared with platelet-rich plasma, or whole blood anti-coagulated with citrate. Thus, whole blood kept at the physiological Ca2+ concentration provides an optimal condition for the formation of PMP. Convulxin, a GPVI-selective agonist, also induced PMP formation at the magnitude which far exceeds those of other agonists, such as thrombin receptor-activating peptide, ADP or epinephrine. These findings suggest that GPVI-mediated platelet activation plays a key role in the formation of PMP in the presence of physiological Ca2+ in whole blood. The addition of red blood cells to PRP potentiated PMP formation induced by collagen. Pretreatment of whole blood with the combination of creatine phosphate and creatine phosphokinase reduced PMP formation induced by collagen. Blockade of ADP receptors, P2Y(12) with AR-C69931MX and P2Y(1) with A3P5P, respectively, further suppressed collagen-induced PMP formation. We conclude that ADP released from red blood cells enhances PMP formation induced by collagen, and that both P2Y(12) and P2Y(1) contribute to ADP-potentiation of PMP generation induced by collagen.