Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pharmacology and Physiology, 2015.
Thromboembolic diseases are major causes of morbidity and mortality. Endothelial exocytosis plays a critical role in thrombosis. The exocytic machinery that regulates granule secretion from endothelial cells (EC) is not completely defined. We hypothesized that specific members of the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) superfamily and their regulatory partners mediate endothelial granule exocytosis and thrombosis.
EC exocytosis releases von Willebrand factor (VWF), a major factor in thrombogenesis. Recent genome-wide association studies (GWAS) identified syntaxin-binding protein 5 (STXBP5) as a candidate gene linked to changes in plasma VWF levels. We found STXBP5 is expressed in human ECs and interacts with SNARE proteins. STXBP5 inhibits endothelial exocytosis in vitro. Stxbp5 KO mice had higher plasma VWF levels and more P-selectin translocation than wild-type mice, suggesting STXBP5 inhibits endothelial exocytosis in vivo. However, Stxbp5 KO mice also displayed impaired hemostasis and thrombosis. Platelets from Stxbp5 KO mice had defects in secretion and activation. Therefore, STXBP5 is a novel SNARE regulatory partner that inhibits endothelial exocytosis, but promotes platelet secretion and thrombosis.
In secretory cells, a ternary complex that mediates exocytosis is comprised of three SNARE molecules, including isoforms of the syntaxin (STX), vesicle-associated membrane protein (VAMP), and synaptosomal-associated protein (SNAP) families.
Human ECs express proteins of the SNARE superfamily, but the exact identity of EC SNAREs that regulate endothelial exocytosis has been unclear. We explored the SNARE molecules required for endothelial exocytosis. We found VAMP8 co-localized with Weibel-Palade bodies, the major endothelial granules. VAMP8 interacted with components of the exocytic machinery. Knock down of VAMP8 expression inhibited endothelial exocytosis. Vamp8 KO mice had decreased endothelial exocytosis. These data suggest that VAMP8 plays a critical role in endothelial exocytosis.
In addition, we identified SNAP23 as the predominant endothelial SNAP isoform that mediates endothelial exocytosis. SNAP23 was localized to the plasma membrane. Knockdown of SNAP23 decreased endothelial exocytosis. SNAP23 also interacted with the endothelial exocytic machinery. These data suggest that SNAP23 is another key component of the endothelial SNARE machinery.
Taken together, the present work identified the role of STXBP5, VAMP8, and SNAP23 in EC exocytosis. Given the importance of EC exocytosis to vascular thrombosis and inflammation, the insights gained from these studies may lead to novel targets for the controlling of thromboembolic diseases.
Howard Hughes Medical Institute (HHMI) - "Med-into-Grad" Fellowship in Cardiovascular Sciences at the Aab Cardiovascular Research Institute, University of Rochester (to Charles J. Lowenstein and Qiuyu Zhu)
