Circulating blood platelets are specialized cells that function to prevent bleeding and minimize blood vessel injury. As such, platelets play a critical role in both normal and disease physiology. The currently favored model of platelet formation states that large progenitor cells in the bone marrow called megakaryocytes (MKs) release platelets by extending long, branching processes, designated proplatelets, into sinusoidal blood vessels. Despite the importance of platelets in thrombosis and hemostasis, the cellular and molecular basis of the process by which MKs complete differentiation and release platelets is poorly understood. In particular, little is known about what triggers resting, mature MKs in the bone marrow to begin forming and releasing proplatelets. Proteomic analysis of resting versus proplatelet-producing MKs suggests that there is a distinct subset of proteins synthesized in the late stages of MK development that are necessary for proplatelet formation. This proposal will focus on two proteins identified by proteomics that are up-regulated in proplatelet-producing MKs, myristoylated, alanine-rich, C kinase substrate (MARCKS) and ubiquilin. Both proteins are involved in cytoskeletal reorganization; MARCKS cross-links F-actin, while ubiquilin links proteins to the proteasome for degradation. I hypothesize that the dynamic cytoskeletal process of proplatelet formation requires both protein synthesis and degradation, and MARCKS and ubiquilin are integral to cytoskeletal remodeling by modulating actin crosslinking and protein degradation, respectively. We will test this hypothesis using techniques such as siRNA, protein overexpression, and immunoprecipitation to establish the role of MARCKS and ubiquilin in proplatelet formation. We will then use the MARCKS knockout mouse and a novel microfluidics system to explore the role of these proteins in an ex vivo environment. The results of the proposed experiments will significantly advance our understanding of the cellular and molecular basis regulating platelet production. Our long-range goal is to elucidate cell biological and molecular pathways that power platelet production, with the intent of defining novel therapeutic strategies to accelerate platelet production in patients with thrombocytopenia.
Platelets are essential for hemostasis; and thrombocytopenia (platelet counts <150x109/L) is a major clinical problem encountered across a number of conditions; including immune thrombocytopenic purpura; chemotherapy; surgery; aplastic anemia; human immunodeficiency virus infection; and complications during pregnancy and delivery. My goal is to identify molecular pathways responsible for proplatelet initiation from ther precursor cells; megakaryocytes; to provide therapeutic targets that can be used to stimulate proplatelet release from existing megakaryocytes; resulting in an immediate increase in platelet count- an ''autotransfusion'' of platelets. Currently; thrombopoeitin administered to thrombocytopenic patients takes 5 days to increase platelet counts and 12 days to reach maximum effect; therefore; a therapy that immediately stimulates platelet production from existing megakaryocytes would be an extremely valuable tool for thrombocytopenia treatment.
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