The purpose of this proposal is to investigate the cell biological and molecular pathways that regulate platelet production. Although it is well established that megakaryocytes generate platelets by remodeling their cytoplasm into proplatelet extensions, which serve as assembly lines for platelet production, many unanswered questions remain regarding the mechanisms of platelet biogenesis. In particular, our understanding of the process by which megakaryocytes initiate proplatelet production is poorly understood. We have recently used a novel microinjection approach to discover a cytoplasmic factor, called proplatelet-promoting factor (PPF) that triggers platelet production when injected into megakaryocytes. Even though our preliminary data have clarified some questions about the basic nature of PPF activity, we only have a cursory understanding of its regulation and mechanism of action. Obtaining this fundamental knowledge is crucial for evaluating the scope of PPF action and determining how it functions in proplatelet initiation. Therefore, in Specific Aim 1 we propose to define the biological nature of PPF activity and isolate PPF so that we can understand how this cytoplasmic factor controls proplatelet induction. Although the mechanism by which the cytoskeleton regulates proplatelet initiation is unknown, the spatial and temporal disassembly of the centrosome after the injection of PPF, but before proplatelet initiation, suggests a role for the centrosome in proplatelet induction. This exciting new data suggests that centrosome disassembly powers induction of proplatelet production, a hypothesis that will be tested in Specific Aim 2. Finally, in Specific Aim 3 we will use a high-content microscopy screen to identify molecules that drive platelet production. Using proplatelet image analysis, we will test thousands of drug molecule candidates for their ability to stimulate or inhibit platelet production. Target pathway analysis, secondary screens, and dose-response curves will be used to identify compound ?hits.? We will then establish whether hits are efficacious in animal models. Taken together, we expect that findings made as a result of this investigation will provide an improved understanding of the molecular mechanisms that regulate platelet formation and lay the foundation for novel therapeutic approaches to accelerate platelet production in patients with thrombocytopenia.
Blood platelets play a critical role in stimulating clot formation and repair of vascular injury. Life-threatening thrombocytopenia (low platelet count) can occur in patients for a variety of reasons, including chemotherapy, radiation treatment, trauma, severe burns, organ transplant surgery, and genetic disorders. The studies proposed in this application will advance our understanding of blood platelet production and may lead to improved therapies for accelerating platelet production in these patients.
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