Megakaryocyte-Derived Microparticles: Mechanism of Formation and Circulation in Plasma.

Abstract Platelet microparticles are submicron membrane vesicles expressing platelet markers and are a normal constituent of circulating blood. Several studies have demonstrated positive correlations between thrombotic states and increased platelet microparticle levels. Yet the physiology of these microparticles is poorly understood. Specifically, the origin of circulating platelet microparticles is not known. Platelet microparticles are routinely formed in vitro following exposure of platelets to pharmacologic concentrations of platelet agonists such as the combination of thrombin and collagen. This observation has lead to the widely held assumption that circulating platelet microparticles are derived from activated platelets. We now have identified and characterized platelet microparticles derived directly from megakaryocytes. Videomicroscopy of microparticle production by mouse megakaryocytes demonstrated an active process in which microparticles form as submicron beads along the lengths of thin, extended pseudopods. These pseudopods were morphologically distinct from the broad, branching cytoplasmic extensions from which proplatelets form. Studies using cytoskeletal inhibitors confirmed that megakaryocytes produce microparticles and platelets by two distinct mechanisms. The microtubule inhibitor nocodazole (5 μM) totally abolished proplatelet formation, but failed to affect microparticle formation. Latrunculin A (5 μM), which depolymerizes F-actin, inhibited proplatelet branching, but resulted in a 2-fold increase in megakaryocyte-derived microparticle formation. Megakaryocyte-derived microparticles had an average diameter of 0.5 μm. These microparticles were CD41+ and bound annexin V, indicated surface phosphatidylserine exposure. To determine whether plasma microparticles in wild-type mice are derived primarily from activated platelets or generated from megakaryocytes, we identified markers that distinguish between these two populations. CD62 and CD63 were found only on microparticles generated by activated platelets. In contrast, full-length filamin A was found in megakaryocyte-derived microparticles, but not in microparticles from activated platelets. Circulating microparticles isolated from mice were CD62−,CD63−, and expressed full-length filamin A indicating a megakaryocytic origin. Similarly, circulating microparticles isolated from healthy volunteers were CD62−,CD63−, and expressed full-length filamin A. These results indicate that direct production by megakaryocytes represents a physiologic means to generate circulating platelet microparticles. We propose that circulating platelet microparticles can be derived from two sources. One source is the mature platelet, from which microparticles are generated following platelet activation in pathological conditions. The second source is megakaryocytes, which produce microparticles in a constitutive manner.