Cytoskeletal Mechanisms of Platelet Formation

描述（由申请方提供）：血小板在止血以及血栓形成的病理生理学中发挥重要作用。本研究的目的是探讨血小板形成的细胞骨架机制。尽管血小板起源于巨核细胞已被公认，但关于血小板形成和释放的机制仍有许多未解之谜。血小板形成的有利模型认识到，终末分化的巨核细胞延伸长的胞质突起，称为前血小板，其在血小板生物发生中起重要的中间结构的作用。我们最近发现了一个新的血小板生成的中间阶段，前血小板，并已确定微管和血影蛋白为基础的膜骨架参与血小板的生产。然而，前血小板如何转化为血小板尚不清楚，细胞骨架对血小板产生的贡献的许多分子细节仍有待揭示。该提案的三个具体目标侧重于细胞骨架在血小板生成中的作用。具体目标1将研究微管如何将preplatelets转化为杠铃形状，从而分裂释放血小板，目的是测试微管滑动为preplatelet分裂和释放提供动力的假设。将建立特定微管相关蛋白的作用。具体目标2将定义血影蛋白为基础的膜骨架如何有助于前血小板的产生。使用新开发的透化前血小板系统，将建立空间和时间定位，以及关键的膜骨架蛋白的功能。将测试细丝蛋白-GPIba-肌动蛋白连接在前血小板向血小板转化中的作用。最后，具体目标3将评价微管在形成分界膜系统中的重要性，目的是使用体外模型来确定分界膜系统是通过在微管上移动的膜相关马达形成的，还是通过附着在伸长微管的尖端形成的。这些实验将确定特定的细胞骨架蛋白在分界膜系统形成中的功能。综上所述，我们期望本研究的结果将提供对调节血小板形成的分子机制的更好理解，并为加速血小板减少症患者血小板生成的新治疗方法以及旨在改善输注用血小板体外生成的策略奠定基础。

DESCRIPTION (provided by applicant): Blood platelets play an essential role in hemostasis, as well as in the pathophysiology of thrombosis. The purpose of this proposal is to investigate the cytoskeletal mechanics of platelet formation. Although it is well established that platelets originate from megakaryocytes, many unanswered questions remain regarding the mechanics by which platelets are formed and released. The favored model of platelet formation recognizes that terminally differentiated megakaryocytes extend long cytoplasmic processes, designated proplatelets, which function as essential intermediate structures in platelet biogenesis. We have recently discovered a new intermediate stage in platelet production, the preplatelet, and have established that microtubules and the spectrin-based membrane skeleton are involved in platelet production. How preplatelets convert into platelets, however, is not known, and many of the molecular details underlying the contribution of the cytoskeleton to platelet production remain to be uncovered. The three Specific Aims of this proposal focus on the role of the cytoskeleton in platelet production. Specific Aim 1 will examine how microtubules convert preplatelets into barbell shapes that divide to release platelets, with the goal of testing the hypothesis that microtubule sliding powers preplatelet fission and release. The role of specific microtubule-associated proteins will be established. Specific Aim 2 will define how the spectrin-based membrane skeleton contributes to proplatelet production. Using a newly developed permeabilized proplatelet system, the spatial and temporal localization, as well as the function of key membrane skeleton proteins will be established. The role of the Filamin-GPIba-actin linkage in the preplatelet to platelet transition will be tested. Finally, Specific Aim 3 will evaluate the importance of microtubules in forming the demarcation membrane system, with the goal of using an in vitro model to determine if the demarcation membrane system forms by membrane- associated motors moving over microtubules or by the attachment to the tips of elongating microtubules. These experiments will define the function of specific cytoskeletal proteins in demarcation membrane system formation. 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, as well as strategies aimed at improving the in vitro generation of platelets for infusion.