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...