Mutations in the MYH9 gene encoding the nonmuscle myosin heavy chain IIA result in bleeding disorders characterized by a macrothrombocytopenia. To understand the role of myosin in normal platelet functions and in pathology, we generated mice with disruption of MYH9 in megakaryocytes. MYH9⌬ mice displayed macrothrombocytopenia with a strong increase in bleeding time and absence of clot retraction. However, platelet aggregation and secretion in response to any agonist were near normal despite absence of initial platelet contraction. By contrast, integrin outside-in signaling was impaired, as observed by a decrease in integrin ␤3 phosphorylation and PtdIns(3,4)P 2 accumulation following stimulation. Upon adhesion on a fibrinogen-coated surface, MYH9⌬ platelets were still able to extend lamellipodia but without stress fiber-like formation. As a consequence, thrombus growth and organization, investigated under flow by perfusing whole blood over collagen, were strongly impaired. Thrombus stability was also decreased in vivo in a model of FeCl 3 -induced injury of carotid arteries. Overall, these results demonstrate that while myosin seems dispensable for aggregation and secretion in suspension, it plays a key role in platelet contractile phenomena and outsidein signaling. These roles of myosin in platelet functions, in addition to thrombocytopenia, account for the strong hemostatic defects observed in MYH9⌬ mice. IntroductionImportant morphologic changes occur in platelets during their activation at sites of vascular injury. The cells lose their resting discoid shape to become spheroid and contracted, emitting membrane blebs and longer extensions. [1][2][3][4] Once in contact with a surface, the spheroid platelets extend long filopodia and finally spread over it by emitting thin, sheet-like lamellipodia. 1,2 Myosin activation plays a central role in the cytoskeletal rearrangements underlying these changes in morphology. Myosin becomes activated after phosphorylation of the myosin regulatory light chain (RLC), which results from both calcium-regulated myosin lightchain kinase activity and Rho kinase-regulated myosin phosphatase activity. [5][6][7][8] Activated myosin assembles into short filaments through the myosin heavy chain and interacts mainly with central actin filaments. Myosin has been proposed to participate in several platelet contractile functions such as platelet spheration, contraction and stress-fiber formation, and fibrin clot retraction. Platelet spheration and contraction, as observed in the aggregometer, closely correlate with phosphorylation of the RLC 9,10 and are prevented when RLC phosphorylation is inhibited. 6,7,9,10 Myosin has also been shown to be associated with stress fiber-like structures in spreading adherent platelets. 11 In addition, myosin could play a role in platelet secretion, as it is decreased by inhibition of myosin RLC phosphorylation. 5,[12][13][14][15] Finally, a role of myosin in clot retraction has long been suspected in view of the necessity for a contractile force and was ...

Key Points• PI3K-C2a controls platelet membrane structure and remodeling.• PI3K-C2a is a key regulator of a basal housekeeping PI3P pool in platelets.The physiologic roles of the class II phosphoinositide 3-kinases (PI3Ks) and their contributions to phosphatidylinositol 3-monophosphate (PI3P) and PI(3,4)P 2 production remain elusive. Here we report that mice heterozygous for a constitutively kinase-dead PI3K-C2a display aberrant platelet morphology with an elevated number of barbell-shaped proplatelets, a recently discovered intermediate stage in the final process of platelet production. Platelets with heterozygous PI3K-C2a inactivation have critical defects in a-granules and membrane structure that are associated with modifications in megakaryocytes. These platelets are more rigid and unable to form filopodia after stimulation. Heterozygous PI3K-C2a inactivation in platelets led to a significant reduction in the basal pool of PI3P and a mislocalization of several membrane skeleton proteins known to control the interactions between the plasma membrane and cytoskeleton. These alterations had repercussions on the performance of platelet responses with delay in the time of arterial occlusion in an in vivo model of thrombosis and defect in thrombus formation in an ex vivo blood flow system. These data uncover a key role for PI3K-C2a activity in the generation of a basal housekeeping PI3P pool and in the control of membrane remodeling, critical for megakaryocytopoiesis and normal platelet production and function. (Blood. 2015;126(9):1128-1137