Structure/Function Determinants of Platelet Granule Secretion

描述（由申请人提供）：α -颗粒是血小板的主要分泌细胞器，含有数百种激活后释放的蛋白质。有趣的是，许多储存的蛋白质似乎具有相反的功能，例如那些具有促进或抗血管生成特性的蛋白质。血小板生理学中一个悬而未决的主要问题是：血小板是一个积极的参与者，特异性地从它们的颗粒中释放出适合环境的物质，还是它们是随机的递送装置？在这里，我们通过三个具体目标来解决这一核心问题的重要方面。具体目标1：验证人类血小板含有单一主要颗粒群的假设，其中单个货物蛋白被包装成不同的区域。通过电子断层扫描、免疫金标记和超分辨率光学显微镜的结合应用，我们将分析整个血小板的颗粒结构和蛋白质分布。使用这些结构方法，我们将确定人类颗粒群体中结构和货物蛋白分布的同质性或异质性的程度。特定目的2：验证特异性颗粒亚域/延伸为响应激动剂的质膜/OCS的差异膜融合/蛋白质分泌提供空间基础的假设。在本研究中，我们应用来自Aim 1的成像方法来表征血小板颗粒分泌支持差异蛋白释放的结构基础。我们的数据和其他人的数据表明，差异释放是颗粒分泌的正常结果。到目前为止，我们的初步数据与一个模型一致，其中重要的融合机制蛋白，如v- SNARE， VAMP-8，集中在¿-颗粒的不同亚结构域上，因此可能介导亚结构域特异性融合。基因敲除的小鼠血小板将有助于揭示颗粒释放中中间体积累的实验。具体目的3：验证VWF和/或细胞骨架元件为血小板颗粒结构和功能提供组织原则的假设。颗粒VWF的可逆解聚具有通过影响蛋白分区和颗粒形状来调节颗粒分泌的治疗潜力。本研究具有重要的意义和创新性。我们的总体假设是，一个颗粒在结构上组织成特定的子域，为激动剂反应性分泌而设计，提供了一个创新的智力框架，推动实验朝着尖锐的答案发展。这个框架可以揭示出其他情况下无法得到的答案。我们在高分辨率成像技术方面的经验为血小板领域带来了一套新的工具集，需要明确回答所提出的核心问题。我们的工作将为未来的治疗设计提供一个参考框架。

DESCRIPTION (provided by applicant): Alpha-granules, the major secretory organelle of platelets, contain hundreds of proteins that are released upon activation. Interestingly, many of the stored proteins have seemingly opposite function, such as those with pro- or anti-angiogenic properties. A major unanswered question in platelet physiology is: Are platelets an active participant specifically releasing context-appropriate material from their ¿-granules or are they random delivery devises? Here we address important aspects of that central question through three Specific Aims. Specific Aim 1: To test the hypothesis that human platelets contain a single major ¿-granule population in which individual cargo proteins are packaged into distinct zones. Through the combined application of electron tomography, immunogold labeling, and super-resolution light microscopy, we will analyze a whole platelet both with respect to granule structure and protein distribution. Using these structural approaches, we will determine the extent of homogeneity, or heterogeneity, in structure and cargo protein distribution in the human ¿-granule population. Specific Aim 2: To test the hypothesis that specialized ¿-granule subdomains/extensions provide a spatial basis for differential membrane fusion/protein secretion to the plasma membrane/OCS in response to agonists. In this Aim, we apply the imaging approaches, from Aim 1, to characterize the structural basis on which platelet ¿-granule secretion can support differential protein release. Our data and that of others suggest that differential release is a normal outcome of ¿-granule secretion. To date, our Preliminary Data are consistent with a model in which important fusion machinery proteins such as the v- SNARE, VAMP-8, are concentrated over distinct subdomains of the ¿-granule and hence may mediate subdomain specific fusion. Mouse platelets from gene knockouts will be facilitate experiments designed to reveal the accumulation of intermediates in granule release. Specific Aim 3: To test the hypothesis that VWF and/or cytoskeletal elements provides an organizing principle for platelet ¿-granule structure and function. Reversible depolymerization of granule VWF has the therapeutic potential to modulate ¿-granule secretion through affecting protein zoning and granule shape. The proposed research is both significant and innovative. Our overarching hypothesis of a granule organized structurally into specific subdomains designed for agonist-responsive secretion provides an innovative intellectual framework that drives experiments towards incisive answers. This framework can lead to revealing answers that would not come otherwise. Our experience in high-resolution imaging technology brings a novel toolset to the platelet field needed to definitively answer the central question raised. Our work will provide a reference framework for future therapeutic design.