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。血小板生理学中一个未解答的主要问题是：血小板是否是专门释放适合环境的物质的积极参与者。从他们的 ¿ -颗粒还是随机输送装置？在这里，我们通过三个具体目标 1 来解决该中心问题的重要方面：检验人类血小板含有单一主要成分的假设。 -颗粒群，其中单个货物蛋白被包装到不同的区域中，通过电子断层扫描、免疫金标记和超分辨率光学显微镜的组合应用，我们将使用这些技术分析整个血小板的颗粒结构和蛋白质分布。结构方法，我们将确定人类结构和货物蛋白分布的同质性或异质性程度 ¿ - 颗粒群体。具体目标 2：检验专门化的假设。 -颗粒子域/扩展为响应激动剂的差异膜融合/蛋白质分泌到质膜/OCS提供空间基础。在这个目标中，我们应用目标1的成像方法来表征血小板的结构基础。 -颗粒分泌可以支持差异蛋白质释放。我们的数据和其他人的数据表明，差异释放是 ¿ 的正常结果。迄今为止，我们的初步数据与一个模型一致，其中重要的融合机器蛋白（例如 v-SNARE、VAMP-8）集中在 ¿ -颗粒，因此可能介导基因敲除的小鼠血小板的亚结构域特异性融合，将有助于旨在揭示颗粒释放中中间体积累的实验。血小板 -颗粒 VWF 的可逆解聚具有调节 ¿ -通过影响蛋白质分区和颗粒形状来实现颗粒分泌。我们提出的关于颗粒在结构上组织成针对激动剂响应性分泌的特定子域的总体假设提供了一个创新的知识框架，可以推动实验获得深入的答案。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，可以明确地回答所提出的核心问题。为未来的治疗设计提供参考框架。