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，指定的前血小板在血小板生物发生中起着必要的中间结构的作用。我们使用小鼠巨核细胞培养系统产生真正的血小板来研究血小板的生物生成过程。血小板的形成始于巨核细胞表面的大伪足的延伸。皮质微管束被用来将这些突起拉长成前血小板，最终在其球茎末端形成突出的微管卷曲。排列在原血小板上的微管的线性阵列也是细胞器和颗粒进入组装的血小板的运输轨迹。肌动蛋白细丝在血小板形成中的重要作用体现在它们对原血小板的放大作用，以及缺乏特定肌动蛋白相关蛋白的血小板的血小板减少和异常形态。尽管我们对血小板生成的了解正在提高，但对细胞骨架促进血小板形成和释放的分子机制知之甚少。针对这一问题，提出了三个具体目标的研究计划。目的1验证微管滑动促进活巨核细胞内血小板延长的假说。分子马达细胞质动力蛋白的贡献将通过击倒和显性-负策略来确定，而原血小板微管阵列的超微结构，包括微管数量、长度和极性将使用电子显微镜进行解析。在目标2中，我们将确定在血小板形成的最后一步中，单个血小板从前血小板释放的机制。这些实验将解决推动最终释放步骤的细胞骨架力量。在最后一个目标中，我们将利用丝氨酸A基因敲除小鼠来研究肌动蛋白结合蛋白丝氨酸A在血小板形成中的作用。我们将使用免疫电子显微镜确定在血小板产生过程中何时建立必要的纤毛素A-血管性血友病因子受体连接。血小板生成和功能的调节是心血管健康的关键决定因素。预计成功完成拟议工作所获得的知识将使我们更好地了解血小板形成的机制，并为止血障碍患者的治疗提供策略建议。

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 that function as essential intermediate structures in platelet biogenesis. We have used a mouse megakaryocyte culture system that produces bonafide platelets to study the process of platelet biogenesis. Platelet formation begins with the extension of large pseudopodia from the megakaryocyte surface. Cortical bundles of microtubules are used to elongate these processes into proplatelets, which ultimately form prominent coils of microtubules at their bulbous ends. The linear arrays of microtubules that line proplatelets also serve as tracks for the transport of organelles and granules into assembling platelets. The importance of actin filaments in platelet formation is demonstrated by their essential role in the amplification of proplatelets and by the thrombocytopenia and abnormal morphology of platelets lacking particular actin associated proteins. Although our understanding of platelet production is improving, little is known of the molecular mechanisms by which the cytoskeleton contributes to platelet formation and release. To address this question, a research plan of three specific aims is proposed. Aim 1 will test the hypothesis that microtubule sliding powers proplatelet elongation in living megakaryocytes. The contribution of the molecular motor cytoplasmic dynein will be established using knock-down and dominant-negative strategies and the ultrastructure of the proplatelet microtubule array, including microtubule numbers, length, and polarity will be resolved using electron microscopy. In Aim 2, we will define the mechanisms by which individual platelets are released from proplatelets during the final step of platelet formation. These experiments will address the cytoskeletal forces that power the final release step. In the last Aim, we will examine the role of the actin binding protein Filamin A in platelet formation using Filamin A knockout mice. We will establish when the essential Filamin A-von Willebrand factor receptor connection is made during platelet production using immunoelectron microscopy. The regulation of platelet production and function are crucial determinants of cardiovascular health. It is anticipated that the knowledge gained by successful completion of the proposed work will provide us with a better understanding of the mechanics of platelet formation and suggest strategies for treatment of patients with disorders of hemostasis.