Identification of molecular pathways that initiate proplatelet formation

描述（由申请人提供）：循环血小板是专门的细胞，其功能是防止出血并最大限度地减少血管损伤。因此，血小板在正常和疾病生理学中起着关键作用。目前流行的血小板形成模型指出，骨髓中称为巨核细胞（MK）的大型祖细胞通过将长的分支过程（称为前血小板）延伸到窦状血管中来释放血小板。尽管血小板在血栓形成和止血中的重要性，但对MK完全分化和释放血小板的过程的细胞和分子基础知之甚少。特别是，很少有人知道是什么触发休息，成熟的MK在骨髓中开始形成和释放前血小板。静息与产生前血小板的MK的蛋白质组学分析表明，在MK发育的后期阶段合成的蛋白质中有一个独特的子集是前血小板形成所必需的。该建议将集中在两个蛋白质组学鉴定的蛋白质，在前血小板生成MK中上调，肉豆蔻酰化，富含丙氨酸，C激酶底物（MARCKS）和泛素。这两种蛋白质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。我推测，动态细胞骨架过程中的前血小板形成需要蛋白质的合成和降解，和MARCKS和泛素是不可或缺的细胞骨架重塑，通过调节肌动蛋白交联和蛋白质降解，分别。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设，以确定MARCKS和泛素在前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离体环境中的作用。拟议的实验结果将显着推进我们的理解的细胞和分子基础调节血小板的生产。我们的长期目标是阐明血小板生成的细胞生物学和分子途径，目的是确定新的治疗策略，以加速血小板减少症患者的血小板生成。

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.