Regulation of endothelial cell phosphatidylserine in thrombosis

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Project Summary/Abstract A prothrombotic vessel wall is implicated as a major driver of cardiovascular disease, including heart attacks, strokes, and venous thromboembolism. Despite extensive research into the mechanisms of endothelial cell dysfunction, the fundamental question of how a clot forms in blood vessels remains incompletely understood. Activation of blood coagulation enzymes requires a membrane surface containing anionic phospholipids, most notably phosphatidylserine (PS). PS-binding proteins decrease thrombosis in animal models suggesting that targeting PS exposure may be a novel antithrombotic strategy. Plasma membrane PS is normally sequestered on the inner membrane leaflet. To activate blood coagulation, PS must be externalized to the outside of the cell by calcium-activated phospholipid scramblases. Platelets readily externalize PS in vitro, and it has often been assumed, but not clearly demonstrated, that platelets generate the procoagulant PS to promote thrombosis in vivo. Our data demonstrate that (1) the majority of PS externalization in vivo actually derives from the vessel wall, independent of platelets, and (2) inhibiting TMEM16 phospholipid scramblases decreases the thrombotic potential of the vessel wall. We have identified two TMEM16 family members, TMEM16F, and its closest paralog TMEM16E, that are both required for PS externalization and procoagulant activity in endothelial cells. TMEM16E is not found in platelets and previously has no known role in coagulation. The central hypothesis of this application is that the endothelial cell membrane is the primary source of PS in forming a thrombus, and its externalization is regulated by two scramblases, TMEM16E and TMEM16F. In Aim 1 we will determine the mechanism by which both TMEM16E and TMEM16F promote PS externalization and procoagulant activity in endothelial cells. In Aim 2 we will use mouse models and intravital microscopy to determine the contribution of endothelial cell-derived PS to thrombosis and how it is regulated by TMEM16E and TMEM16F in vivo. This proposal describes a five-year research training program for Dr. Alec Schmaier’s mentored career development. The applicant has a strong research background in platelet activation from the Medical Scientist Training Program at the University of Pennsylvania. This proposal expands his skill set through investigations in endothelial cell biology, blood coagulation and vessel wall thrombosis. Dr. Robert Flaumenhaft in the Division of Hemostasis and Thrombosis at Beth Israel Deaconess Medical Center will be the primary research mentor. Dr. Flaumenhaft has a distinguished record of mentorship and innovation in thrombosis research. In addition, the applicant’s advisory committee will provide highly relevant scientific expertise and critical assessment of his progress. In sum, Dr. Schmaier has created an outstanding environment to advance his research and career goals to be a physician-scientist in cardiovascular medicine. This comprehensive training program will position him to succeed as an independent investigator in thrombosis and vascular biology.