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• Myosin IIA deficiency affects F-actin structuration and organelle distribution in MKs which leads to abnormal platelet organelle content.During proplatelet formation, a relatively homogeneous content of organelles is transported from the megakaryocyte (MK) to the nascent platelets along microtubule tracks. We found that platelets from Myh9 2/2 mice and a MYH9-RD patient were heterogeneous in their organelle content (granules and mitochondria). In addition, Myh9 2/2 MKs have an abnormal cytoplasmic clustering of organelles, suggesting that the platelet defect originates in the MKs. Myosin is not involved in the latest stage of organelle traffic along microtubular tracks in the proplatelet shafts as shown by confocal observations of proplatelet buds. By contrast, it is required for the earlier distribution of organelles within the large MK preplatelet fragments shed into the sinusoid circulation before terminal proplatelet remodeling. We show here that F-actin is abnormally clustered in the cytoplasm of Myh9 2/2 MKs and actin polymerization is impaired in platelets. Myosin IIA is required for normal granule motility and positioning within MKs, mechanisms that may be dependent on organelle traveling and tethering onto F-actin cytoskeleton tracks. Altogether, our results indicate that the distribution of organelles within platelets critically depends on a homogeneous organelle distribution within MKs and preplatelet fragments, which requires myosin IIA. (Blood. 2014;123(8):1261-1269