Identification of molecular pathways that initiate proplatelet formation

说明(申请人提供)：循环中的血小板是一种特殊的细胞，其功能是防止出血和最大限度地减少血管损伤。因此，血小板在正常生理和疾病生理中都起着至关重要的作用。目前流行的血小板形成模型表明，骨髓中被称为巨核细胞(MKs)的大祖细胞通过将被称为前血小板的长分支过程延伸到正弦血管中来释放血小板。尽管血小板在血栓形成和止血中具有重要作用，但MKs完全分化和释放血小板的细胞和分子基础尚不清楚。特别是，关于是什么触发骨髓中静息的、成熟的巨噬细胞开始形成和释放前血小板，人们知之甚少。对静息和产生原血小板的巨噬细胞的蛋白质组学分析表明，在巨核细胞发育的后期合成了一组独特的蛋白质，这是形成原血小板所必需的。这项建议将集中在蛋白质组学确定的两种蛋白质，它们是在产生血小板的MKs中上调的肉豆蔻酰化的富含丙氨酸的C激酶底物(MARCKS)和泛素蛋白。这两种蛋白质都参与了细胞骨架的重组；Marcks使F-肌动蛋白交叉连接，而泛素则将蛋白质连接到蛋白酶体以进行降解。我假设，原血小板形成的动态细胞骨架过程既需要蛋白质的合成，也需要蛋白质的降解，Marcks和泛素分别通过调节肌动蛋白交联和蛋白质降解而参与细胞骨架的重塑。我们将使用siRNA、蛋白质过表达和免疫沉淀等技术来验证这一假说，以确定Marcks和泛素在原血小板形成中的作用。然后，我们将使用Marcks基因敲除小鼠和一种新的微流体系统来探索这些蛋白质在体外环境中的作用。这些实验的结果将极大地促进我们对调节血小板生成的细胞和分子基础的理解。我们的长期目标是阐明促进血小板产生的细胞生物学和分子途径，目的是定义新的治疗策略来加速血小板减少患者的血小板产生。

DESCRIPTION (provided by applicant): 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.