Regular Article PLATELETS AND THROMBOPOIESIS Q:A1 Myosin-II repression favors pre/proplatelets but shear activation generates platelets and fails in macrothrombocytopenia

• Myosin-II inhibition (with blebbistatin) and MYH9-RD mutations enhance shear fragmentation to pre/ proplatelet sizes. • Sustained shear activates normal myosin-II, which then favors division of pre/ proplatelets to smaller platelets. Megakaryocyte ploidy and the generation of pre/proplatelets are both increased in culture by pharmacologic inhibition of myosin-II, but nonmuscle myosin-IIA (MIIA) mutations paradoxically cause MYH9-related diseases (MYH9-RD) that adversely affect platelets. In marrow, megakaryocytes extend projections into the microcirculation, where shear facilitates fragmentation to large pre/proplatelets, suggesting that fluid stresses and myosin-II activity somehow couple in platelet biogenesis. Here, in bulk shear, plateletlike particles generated from megakaryocytes are maximized at a shear stress typical of that in the microcirculation andafter treatmentwith amyosin-II inhibitor.MIIA activity in static cells is naturally repressed through phosphorylation at Serine-1943, but shear decreases phosphorylation, consistent withMIIA activation and localization to platelet cortex. Micropipette aspiration of cells shows myosin-II accumulates at stressed sites, but its inhibition prevents such mechanoactivation and facilitates generation of CD41 fragments similar in size to pre/proplatelets. MYH9-RD mutants phenocopy inhibition, revealing a dominant negative effect. MIIA is diffuse in the large platelets of aMYH9-RD patient with macrothrombocytopenia and is also diffuse in normal pre/proplatelets treated with inhibitor that blocks in vitro division to small platelets. The findings explain the large platelets inMYH9-RD and the near-normal thrombocrit of patients. Myosin-II regulation thus controls platelet size and number. (Blood. 2014;00(00):1-9)