Structure/Function Determinants of Platelet Granule Secretion

血小板颗粒分泌的结构/功能决定因素

• 批准号: 8562359
• 负责人: Brian Storrie
• 金额: $ 33.28万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8562359
• 关键词: Accounting; Address; Affect; Agonist; Alpha Granule; Anabolism; Blood Platelets; Blood Vessels; Bolus Infusion; Cell membrane; Cellular biology; Chimeric Proteins; Cytoplasmic Granules; Data; Development; Devices; Elements; Endothelial Cells; Fibrinogen; Fluorescence Microscopy; Future; Health; Hemorrhage; Hemostatic function; Heterogeneity; Human; Image; Imaging technology; Individual; Label; Lead; Mediating; Membrane Fusion; Modeling; Mus; Organelles; Outcome; Participant; Physiological; Physiology; Population; Process; Property; Protein Secretion; Proteins; Research; Resolution; Rest; Shapes; Spatial Distribution; Stimulus; Stroke; Structure; Sum; System; Testing; Therapeutic; Thrombosis; Time; Tubular formation; Vascular Diseases; Weibel-Palade Bodies; Work; base; depolymerization; design; electron tomography; experience; fluorescence imaging; innovation; knockout gene; light microscopy; novel; novel therapeutic intervention; protein distribution; public health relevance; research study; response; vesicular SNARE proteins; von Willebrand Factor

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.

描述（由申请人提供）：α-颗粒是血小板的主要分泌细胞器，含有数百种在激活后释放的蛋白质。有趣的是，许多储存的蛋白质具有看似相反的功能，例如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解答的主要问题是：血小板是一个主动参与者，专门从其颗粒中释放适合环境的物质，还是随机输送装置？在这里，我们通过三个具体目标来解决该中心问题的重要方面。具体目标 1：检验人类血小板含有单一主要 ¿-颗粒群的假设，其中各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程度。具体目标 2：检验以下假设：专门的 ¿-颗粒子结构域/扩展为响应激动剂而向质膜/OCS 进行差异膜融合/蛋白质分泌提供空间基础。在此目标中，我们应用目标 1 中的成像方法来表征血小板 ¿-颗粒分泌可以支持差异蛋白质释放的结构基础。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。迄今为止，我们的初步数据与模型一致，其中重要的融合机器蛋白（例如 v-SNARE、VAMP-8）集中在 ¿-颗粒的不同子域上，因此可能介导子域特异性融合。基因敲除的小鼠血小板将有助于旨在揭示颗粒释放过程中中间体积累的实验。具体目标 3：检验 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质分区和颗粒形状来调节 ¿-颗粒分泌的治疗潜力。拟议的研究既重要又具有创新性。我们的总体假设是，颗粒在结构上组织成特定的子域，设计用于激动剂响应性分泌，提供了一个创新的知识框架，推动实验获得深入的答案。这个框架可以揭示其他方式无法得出的答案。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颖的工具，可以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参考框架。