Quantitation of microparticles released from coated‐platelets

Dual agonist stimulation of platelets with thrombin and convulxin results in generation of coated‐platelets, a sub‐population of cells known formerly as COAT‐platelets (collagen and thrombin). Coated‐platelets retain several procoagulant proteins on their surface and express phosphatidylserine (PS). In this report, we utilize a new methodology to demonstrate that coated‐platelets also release microparticles. Platelets were prelabeled with 2.5 μm Bodipy‐maleimide and then stimulated with convulxin plus thrombin. Microparticles, 0.3–0.5 μm in diameter, were observed by fluorescence confocal microscopy. Confocal microscopy was also used to demonstrate that microparticles were positive for glycoprotein IIb/IIIa, glycoprotein Ib, CD9, and PS, but negative for fibrinogen and thrombospondin. Furthermore, microparticles released from Bodipy‐labeled platelets were observed by flow cytometry, and activation with convulxin plus thrombin produced 15 ± 5 microparticles per coated‐platelet. In contrast, platelets stimulated with thrombin or convulxin alone produced few microparticles. Phenylarsine oxide and diamide, both of which potentiate the mitochondrial permeability transition pore and coated‐platelet production, significantly increased the number of microparticles released per coated‐platelet.