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中起着至关重要的作用。本提案的目的是研究血小板形成的细胞骨架力学。虽然血小板起源于巨核细胞已被证实，但关于血小板形成和释放的机制仍有许多未解之谜。最受欢迎的血小板形成模型认为，终末分化的巨核细胞延长了很长的细胞质过程，称为原血小板，它在血小板生物形成中起重要的中间结构作用。我们最近发现了血小板生成的一个新的中间阶段，血小板前期，并确定了微管和基于光谱的膜骨架参与血小板生成。然而，预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揭示。这一建议的三个具体目标集中在细胞骨架在血小板产生中的作用。具体目标1将研究微管如何将血小板前体转化为杠铃形状，并分裂释放血小板，目的是测试微管滑动推动血小板前体裂变和释放的假设。特异性微管相关蛋白的作用将被确定。特异性目标2将定义基于光谱的膜骨架如何促进血小板生成。利用新开发的渗透性血小板系统，建立关键膜骨架蛋白的时空定位和功能。将测试丝蛋白- gpiba -肌动蛋白连锁在血小板前向血小板过渡中的作用。最后，具体目标3将评估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，还是通过附着在延长的微管尖端形成的。这些实验将确定特定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理解，并为加速血小板减少患者血小板产生的新治疗方法奠定基础，以及旨在改善输注血小板体外生成的策略。

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.