Cytoskeletal Mechanisms of Platelet Formation

该提案的目的是研究细胞生物学和分子途径， 调节血小板生成。虽然巨核细胞产生 血小板通过重塑他们的细胞质成为前血小板的延伸，这是作为组装 血小板生产线，许多未回答的问题仍然是关于机制， 血小板生物发生特别是，我们对巨核细胞 对引发前血小板产生知之甚少。我们最近用一本小说 显微注射方法发现一种细胞质因子，称为前血小板促进因子 (PPF)当注射到巨核细胞时会触发血小板的产生。即使我们的 初步的数据已经澄清了一些关于PPF活动的基本性质的问题，我们只 对它的调节和作用机制有一个粗略的了解。获得该 基础知识对于评估PPF行动的范围和确定如何 在前血小板启动中起作用。因此，在具体目标1中，我们建议定义 PPF活性的生物学性质和分离PPF，以便我们能够了解 细胞质因子控制前血小板诱导。虽然这种机制 细胞骨架调节血小板前体的起始是未知的， 注射PPF后，但在血小板前体启动前，中心体的作用表明， 中心体在前血小板诱导中的作用。这个令人兴奋的新数据表明， 拆卸功率诱导前血小板生产，这一假设将在 具体目标2。最后，在具体目标3中，我们将使用高内容显微镜屏幕， 识别驱动血小板生成的分子。使用前血小板图像分析，我们将测试 数千种药物分子候选物，因为它们具有刺激或抑制血小板产生的能力。 靶向途径分析、二次筛选和剂量反应曲线将用于识别 复合"命中"。然后我们将确定命中是否在动物模型中有效。采取 总之，我们希望这次调查的结果将提供一个更好的 了解调节血小板形成的分子机制， 新的治疗方法的基础，以加速血小板生成的患者 血小板减少症

The purpose of this proposal is to investigate the cell biological and molecular pathways that regulate platelet production. Although it is well established that megakaryocytes generate platelets by remodeling their cytoplasm into proplatelet extensions, which serve as assembly lines for platelet production, many unanswered questions remain regarding the mechanisms of platelet biogenesis. In particular, our understanding of the process by which megakaryocytes initiate proplatelet production is poorly understood. We have recently used a novel microinjection approach to discover a cytoplasmic factor, called proplatelet-promoting factor (PPF) that triggers platelet production when injected into megakaryocytes. Even though our preliminary data have clarified some questions about the basic nature of PPF activity, we only have a cursory understanding of its regulation and mechanism of action. Obtaining this fundamental knowledge is crucial for evaluating the scope of PPF action and determining how it functions in proplatelet initiation. Therefore, in Specific Aim 1 we propose to define the biological nature of PPF activity and isolate PPF so that we can understand how this cytoplasmic factor controls proplatelet induction. Although the mechanism by which the cytoskeleton regulates proplatelet initiation is unknown, the spatial and temporal disassembly of the centrosome after the injection of PPF, but before proplatelet initiation, suggests a role for the centrosome in proplatelet induction. This exciting new data suggests that centrosome disassembly powers induction of proplatelet production, a hypothesis that will be tested in Specific Aim 2. Finally, in Specific Aim 3 we will use a high-content microscopy screen to identify molecules that drive platelet production. Using proplatelet image analysis, we will test thousands of drug molecule candidates for their ability to stimulate or inhibit platelet production. Target pathway analysis, secondary screens, and dose-response curves will be used to identify compound “hits.” We will then establish whether hits are efficacious in animal models. 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.