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: 8707849
• 负责人: Brian Storrie
• 金额: $ 36.49万
• 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/8707849
• 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（由适用提供）：血小板的主要秘密细胞器Alpha-Granules包含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，例如具有促血管生成特性的蛋白质。血小板生理学中的一个主要问题是：血小板是一个活跃的参与者，专门从其粒子中释放出适合上下文的材料，还是它们随机递送设计？在这里，我们通过三个特定目标解决了该中心问题的重要方面。特定目的1：测试人血小板包含单个主要„的假设，其中单个货物蛋白包装成不同的区域。通过电子断层扫描，免疫质量标记和超分辨率光学显微镜的联合应用，我们将在颗粒结构和蛋白质分布方面分析整个血小板。使用这些结构方法，我们将确定人类种群中的结构和货物蛋白分布的同质性或异质性的程度。具体目的2：检验以下假设：晶粒子域/扩展为响应激动剂的差异膜融合/蛋白质分泌提供了差异膜融合/蛋白质分泌的空间基础。在此目标中，我们将成像方法（从AIM 1）应用到表征血小板分泌可以支持差异蛋白质释放的结构基础。我们的数据和其他数据表明，差异释放是晶体分泌的正常结果。迄今为止，我们的初步数据与重要的融合机械蛋白（如V- SNARE，VAMP-8）集中在晶状体的不同亚域上，因此可能介导子域特定融合的模型一致。基因敲除的小鼠血小板将是准备旨在揭示颗粒释放中中间体的积累的实验。特定目的3：检验VWF和/或细胞骨架元素为血小板结构和功能提供的组织原理的假设。颗粒VWF的可逆沉积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创新。我们对颗粒的总体假设在结构上组织成专为激动剂反应性分泌而设计的特定子域，这提供了一个创新的智力框架，可将实验推向敏锐的答案。这个框架可能会导致揭示其他不会出现的答案。我们在高分辨率成像技术方面的经验将新颖的工具集带到了确切地回答提出的核心问题所需的血小板领域。我们的工作将为未来的热设计提供参考框架。