Cytoskeletal Mechanisms of Platelet Formation

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DESCRIPTION (provided by applicant): Blood platelets play an essential role in hemostasis, as well as in the pathophysiology of thrombosis. The purpose of this proposal is to investigate the cytoskeletal mechanics of platelet formation. Although it is well established that platelets originate from megakaryocytes, many unanswered questions remain regarding the mechanics by which platelets are formed and released. The favored model of platelet formation recognizes that terminally differentiated megakaryocytes extend long cytoplasmic processes, designated proplatelets that function as essential intermediate structures in platelet biogenesis. We have used a mouse megakaryocyte culture system that produces bonafide platelets to study the process of platelet biogenesis. Platelet formation begins with the extension of large pseudopodia from the megakaryocyte surface. Cortical bundles of microtubules are used to elongate these processes into proplatelets, which ultimately form prominent coils of microtubules at their bulbous ends. The linear arrays of microtubules that line proplatelets also serve as tracks for the transport of organelles and granules into assembling platelets. The importance of actin filaments in platelet formation is demonstrated by their essential role in the amplification of proplatelets and by the thrombocytopenia and abnormal morphology of platelets lacking particular actin associated proteins. Although our understanding of platelet production is improving, little is known of the molecular mechanisms by which the cytoskeleton contributes to platelet formation and release. To address this question, a research plan of three specific aims is proposed. Aim 1 will test the hypothesis that microtubule sliding powers proplatelet elongation in living megakaryocytes. The contribution of the molecular motor cytoplasmic dynein will be established using knock-down and dominant-negative strategies and the ultrastructure of the proplatelet microtubule array, including microtubule numbers, length, and polarity will be resolved using electron microscopy. In Aim 2, we will define the mechanisms by which individual platelets are released from proplatelets during the final step of platelet formation. These experiments will address the cytoskeletal forces that power the final release step. In the last Aim, we will examine the role of the actin binding protein Filamin A in platelet formation using Filamin A knockout mice. We will establish when the essential Filamin A-von Willebrand factor receptor connection is made during platelet production using immunoelectron microscopy. The regulation of platelet production and function are crucial determinants of cardiovascular health. It is anticipated that the knowledge gained by successful completion of the proposed work will provide us with a better understanding of the mechanics of platelet formation and suggest strategies for treatment of patients with disorders of hemostasis.