Structure/Function Determinants of Platelet Granule Secretion

描述(申请人提供)：α颗粒，血小板的主要分泌细胞器，含有数百种蛋白质，在激活时释放。有趣的是，许多储存的蛋白质似乎具有相反的功能，例如那些具有促血管生成或抗血管生成特性的蛋白质。血小板生理学中一个尚未回答的主要问题是：血小板是一种主动参与者，专门从其颗粒中释放与上下文相关的物质，还是随机递送装置？在这里，我们通过三个具体目标处理这一中心问题的重要方面。具体目标1：检验人类血小板包含单个主要颗粒群体的假设，在该群体中，单个货物蛋白被包装到不同的区域。通过电子断层扫描、免疫金标记法和超分辨光学显微镜的联合应用，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种群在结构和货物蛋白分布上的同质性或异质性的程度。具体目标2：验证这样一种假设，即特殊的颗粒亚域/延伸为不同的膜融合/蛋白分泌到质膜/OCS响应激动剂提供了空间基础。在这个目标中，我们应用成像方法，从目标1，来描述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是一致的，在该模型中，重要的融合机械蛋白，如v-SNARE，VAMP-8，集中在不同的亚区颗粒上，因此可能介导亚区特异性融合。从小鼠血小板中剔除基因将有助于实验设计，以揭示中间产物在颗粒中的累积释放。具体目标3：验证VWF和/或细胞骨架元素为血小板颗粒结构和功能提供组织原理的假设。VWF颗粒的可逆解聚可能通过影响蛋白质区带和颗粒形状来调节颗粒的分泌。本文提出的研究具有重要意义和创新性。我们的总体假设是，颗粒在结构上组织成特定的亚域，专为激动剂反应分泌而设计，提供了一个创新的智力框架，推动实验走向尖锐的答案。这一框架可能导致揭示否则不会出现的答案。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，需要它来明确回答提出的中心问题。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.