Identification of molecular pathways that initiate proplatelet formation

描述（由申请人提供）：循环血小板是一种特殊的细胞，具有防止出血和减少血管损伤的功能。因此，血小板在正常和疾病生理中都起着至关重要的作用。目前最受青睐的血小板形成模型认为，骨髓中被称为巨核细胞的大祖细胞（mk）通过延长长分支过程释放血小板，称为原血小板，进入窦状血管。尽管血小板在血栓形成和止血中的重要性，但mk完成分化和释放血小板过程的细胞和分子基础尚不清楚。特别是，对于是什么触发了骨髓中静止的、成熟的mk开始形成和释放原血小板，我们知之甚少。静息与产生血小板的MK的蛋白质组学分析表明，在MK发育的后期，有一种独特的蛋白质亚群是形成血小板所必需的。该提案将重点关注蛋白质组学鉴定的两种蛋白，它们在产生血小板的mk中上调，肉豆蔻酰基化，富含丙氨酸，C激酶底物（MARCKS）和泛素。这两种蛋白都参与细胞骨架重组；MARCKS交联f -肌动蛋白，而泛素将蛋白质连接到蛋白酶体进行降解。我假设血小板形成的动态细胞骨架过程需要蛋白质合成和降解，而MARCKS和泛素分别通过调节肌动蛋白交联和蛋白质降解来参与细胞骨架重塑。我们将使用siRNA、蛋白过表达和免疫沉淀等技术来验证这一假设，以确定marks和泛素在血小板形成中的作用。然后，我们将使用MARCKS敲除小鼠和一种新型微流体系统来探索这些蛋白质在离体环境中的作用。所提出的实验结果将显著促进我们对调节血小板产生的细胞和分子基础的理解。我们的长期目标是阐明驱动血小板产生的细胞生物学和分子途径，以确定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.