Regulation of endothelial cell phosphatidylserine in thrombosis

项目概要/摘要 促血栓形成的血管壁是心血管疾病的主要驱动因素，包括心脏病发作、 中风和静脉血栓栓塞。尽管对内皮细胞的机制进行了广泛的研究 功能障碍，血管中凝块如何形成的基本问题仍不完全清楚。 凝血酶的激活需要含有阴离子磷脂的膜表面，大多数 尤其是磷脂酰丝氨酸（PS）。 PS 结合蛋白可减少动物模型中的血栓形成，表明 针对PS暴露可能是一种新颖的抗血栓策略。质膜 PS 通常是隔离的 在内膜小叶上。为了激活血液凝固，PS必须外化到细胞外部 通过钙激活的磷脂混杂酶。血小板很容易在体外将 PS 外化，并且经常被 假设但未明确证明，血小板产生促凝血 PS 来促进血栓形成 体内。我们的数据表明 (1) 体内大部分 PS 外化实际上来自血管 壁，独立于血小板，并且（2）抑制 TMEM16 磷脂扰乱酶可减少血栓形成 血管壁的电位。我们已经确定了两个 TMEM16 家族成员 TMEM16F 及其最接近的成员 旁系同源物 TMEM16E，是内皮细胞 PS 外化和促凝血活性所必需的。 TMEM16E 在血小板中未发现，并且之前在凝血中的作用尚不清楚。中心假设为 该应用认为内皮细胞膜是形成血栓的PS的主要来源，其 外化由两个扰乱酶 TMEM16E 和 TMEM16F 调节。在目标 1 中，我们将确定 TMEM16E 和 TMEM16F 促进 PS 外化和促凝血活性的机制 内皮细胞。在目标 2 中，我们将使用小鼠模型和活体显微镜来确定 内皮细胞来源的 PS 与血栓形成的关系及其在体内如何受 TMEM16E 和 TMEM16F 的调节。 该提案描述了 Alec Schmaier 博士指导职业生涯的五年研究培训计划 发展。申请人拥有医学科学家在血小板活化方面深厚的研究背景 宾夕法尼亚大学的培训计划。该提案通过调查扩展了他的技能 内皮细胞生物学、凝血和血管壁血栓形成。罗伯特·弗劳门哈夫特博士 贝斯以色列女执事医疗中心的止血和血栓科将是主要研究对象 导师。 Flaumenhaft 博士在血栓研究领域的指导和创新方面拥有杰出的记录。在 此外，申请人的咨询委员会将提供高度相关的科学专业知识和关键的 评估他的进步。总之，施迈尔博士为他的发展创造了良好的环境。 成为心血管医学领域的医师科学家的研究和职业目标。此次综合培训 该计划将使他成为血栓形成和血管生物学领域的独立研究者。

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.