Molecular Mechanisms Underlying Endothelial Weibel-Palade Body Biogenesis and Exocytosis

项目摘要/摘要 血管内皮细胞在止血和血栓形成中起着至关重要的作用。保持血液流动性是必不可少的，但它也 表达并释放大量调节血细胞活化和凝血的“因子”。其中， 血管性血友病因子(von Willebrand factor，vWF)是血管内皮细胞释放的一种重要的血浆止血因子。缺憾 成熟的vWF是人类最常见的出血性疾病。另一方面，血浆中高水平的 VWF，或其高分子量多聚体的异常浓度，与增加的风险有关 心血管疾病发病率。因此，对调节释放的分子机制的理解 VWF在止血和血栓形成方面具有广泛的意义。一个对正常生活起着关键作用的因素 内皮细胞的VWF胞吐作用不影响其储存颗粒的生物发生 身体(WPB)，这是一个高度复杂的过程，仍然没有得到很好的描述。我们之前已经 研究表明，溶酶体相关细胞器复合体2(BLOC-2)的生物发生缺陷会导致 VWF胞吐。因为，BLOC-2和其他相关的运输蛋白在生物发生中是必不可少的 溶酶体相关细胞器，一组特殊颗粒，包括血小板致密颗粒和 黑素小体，这种蛋白质复合体似乎也是WPB生物发生所必需的。我们 假设BLOC-2介导的内体转运对WPB的生物发生至关重要。我们进一步 假设外囊复合体通过1)与BLOC-2相互作用在WPB的运输中发挥重要作用 它在内体转运和调节可溶性NSF接头蛋白受体(SNARE)介导中的作用 白细胞在质膜上的融合。在目标1中，我们将描述来自内体的货物贩运的特征 与成熟的WPBs和该途径对BLOC-2的依赖有关。在目标2中，我们将评估 依赖BLOC-2的内体转运中的胞囊复合体及其核心转运的特征 参与这一途径的机械。最后，在目标3中，我们将评估 圈套介导的WPBs在质膜融合中的胞囊复合体。 申请者Anish Sharda博士之前完成了血液学的临床和研究奖学金 目前正在Robert Flaumenhaft博士的指导下进行博士后研究 布鲁斯·弗里，他们将分别担任主要导师和共同导师。止血与止血分科 贝丝以色列女执事医疗中心的血栓形成在科学创新方面有着杰出的记录 并将为申请者追求其职业目标提供一个良好的环境。这个 Sharda博士建立的杰出科学家顾问委员会将带来不同的知识分子 为他的训练和科学成长提供专业知识。Sharda博士完全有资格执行拟议的实验 并提出了一个全面的五年计划，以实现他成为独立研究员的目标。

Project Summary/Abstract Endothelium plays a vital role in hemostasis and thrombosis. It is essential to maintain blood fluidity, but it also expresses and releases numerous “factors” that regulate blood cell activation and coagulation. Among these, von Willebrand factor (vWF) is an essential plasma hemostatic factor released by the endothelium. Deficiency of mature vWF is the most common bleeding disorder in humans. On the other hand, elevated plasma levels of vWF, or abnormal concentrations of its high-molecular weight multimers, is associated with increased risk of cardiovascular morbidity. As such, understanding of the molecular mechanisms that underlie regulated release of vWF have broad implications in hemostasis and thrombosis. One factor that plays a crucial role in normal vWF exocytosis from endothelial cells is unimpaired biogenesis of its storage granules, the Weibel-Palade bodies (WPBs), which is a highly complex process that remains poorly characterized. We have previously shown that deficiency of biogenesis of lysosome-related organelle complex 2 (BLOC-2) results in impaired vWF exocytosis. Since, BLOC-2, and other related trafficking proteins, are essential for biogenesis of lysosomal-related organelles, a group of specialized granules that includes platelet dense granules and melanosomes, it seems plausible that this protein complex is also required for biogenesis of WPB. We hypothesize that BLOC-2-mediated endosomal trafficking is critical for biogenesis of WPB. We further postulate that the exocyst complex plays an essential role in WPB trafficking by 1) interacting with BLOC-2 in its role in endosomal trafficking 2) and regulating soluble NSF adaptor protein receptor (SNARE)-mediated fusion of WPBs at the plasma membrane. In Aim 1, we will characterize cargo trafficking from the endosomes to the maturing WPBs and the dependence of this pathway on BLOC-2. In Aim 2, we will evaluate the role of the exocyst complex in BLOC-2-dependent endosomal trafficking and characterize the core trafficking machinery involved in this pathway. And, finally, in Aim 3, we will evaluate the regulatory function of the exocyst complex in SNARE-mediated fusion of WPBs at the plasma membrane. The applicant, Dr. Anish Sharda, previously completed clinical and research fellowship in hematology and is currently pursuing a post-doctoral fellowship under the mentorship of Drs. Robert Flaumenhaft and Bruce Furie, who will serve as primary mentor and co-mentor, respectively. The Division of Hemostasis and Thrombosis at Beth Israel Deaconess Medical Center has a distinguished track record of scientific innovation and mentorship, and will provide an excellent environment for the applicant in pursuit of his career goals. The advisory committee of exceptional scientists that Dr. Sharda has established will bring diverse intellectual expertise to his training and scientific growth. Dr. Sharda is well-qualified to execute the proposed experiments and has presented a comprehensive five-year plan to meet his goal of becoming an independent researcher.