Cytoskeletal Mechanisms of Platelet Formation

描述(申请人提供)：血小板在止血和血栓形成的病理生理学中起着重要的作用。这项建议的目的是研究血小板形成的细胞骨架机制。尽管众所周知，血小板起源于巨核细胞，但关于血小板形成和释放的机制仍有许多悬而未决的问题。最受欢迎的血小板形成模型认为，终末分化的巨核细胞延伸了较长的细胞质突起，称为前血小板，在血小板生物发生中起着必不可少的中间结构的作用。我们最近发现了血小板产生的一个新的中间阶段，即血小板前阶段，并证实微管和基于血影蛋白的膜骨架参与了血小板的产生。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背后的许多分子细节仍未被发现。这项提案的三个具体目标集中在细胞骨架在血小板生产中的作用。具体目标1将研究微管如何将前血小板转化为杠铃形状，再分裂为释放血小板，目的是验证微管滑动增强前血小板分裂和释放的假说。特定的微管相关蛋白的作用将被确定。具体目标2将定义以血影蛋白为基础的膜骨架如何促进原血小板的产生。利用新开发的渗透性原血小板系统，将建立关键的膜骨架蛋白的空间和时间定位以及功能。丝氨酸-GPIba-肌动蛋白连接在前血小板到血小板转变中的作用将被测试。最后，具体目标3将评估微管在形成定界膜系统中的重要性，目标是使用体外模型来确定定界膜系统是通过膜相关马达在微管上移动还是通过附着在细长的微管末端形成的。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，我们期待这项研究的结果将提供对调节血小板形成的分子机制的更好的理解，并为促进血小板减少症患者的血小板产生的新的治疗方法以及旨在改善用于输注的血小板的体外产生的策略奠定基础。

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.