Structure and Function of Protein-Membrane Interactions in Blood Clotting

描述(申请人提供)：血液凝固的几乎每一步都需要在膜表面组装多种蛋白质，但在血液凝固过程中蛋白质-膜的相互作用在分子水平上仍然知之甚少。我们正在使用包括魔角旋转固态核磁共振(SS核磁共振)在内的新的高分辨率技术来探索凝血蛋白与磷脂表面相互作用的机制，以及膜组成的变化如何调节凝血反应。我们研究的主要焦点是组织因子和因子VIIa的膜结合复合体，这种两个亚单位的酶在健康和疾病中负责触发血液凝结。目的1将在原子分辨率下描述在钙离子存在下富PS膜结构和动力学的变化，并将研究磷脂酰丝氨酸、磷脂酰胆碱和磷脂酰乙醇胺在配置膜以支持凝血因子高亲和力结合中的作用。目的2研究磷脂-磷脂相互作用在促进膜表面凝血反应中的作用。目标3将描述凝血蛋白与双层结合时诱导的脂质环境/构象。目的4解决组织因子在膜表面的结构，并确定其与配体相互作用时的构象变化。总而言之，这些研究将在原子尺度上为膜表面在血液凝结中的作用提供有价值的新见解。 公共卫生相关性：在美国，凝血系统紊乱是导致残疾和死亡的主要原因，但我们对分子水平上的凝血反应仍有非常不完整的了解。这些研究将为血液凝结系统的调节提供新的线索，尤其是对如何以及为什么在膜表面发生血液凝结反应的详细了解。

DESCRIPTION (provided by applicant): Almost every step in blood coagulation requires assembly of multiple proteins on membrane surfaces, yet protein-membrane interactions in blood clotting remain poorly understood at the molecular level. We are using new, high-resolution technologies including magic-angle spinning solid-state NMR (SSNMR) to probe the mechanisms by which blood clotting proteins interact with phospholipid surfaces, and how changes in membrane composition regulate clotting reactions. The primary focus of our studies is the membrane-bound complex of tissue factor and factor VIIa, the two-subunit enzyme responsible for triggering blood clotting in health and disease. Aim 1 will delineate, at atomic resolution, the changes in structure and dynamics of PS- rich membrane domains in the presence of Ca2+, and will examine the roles of phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine in configuring the membrane to support high affinity binding of clotting factors. Aim 2 will investigate the role of phospholipid-phospholipid interactions in enhancing blood clotting reactions on membrane surfaces. Aim 3 will delineate the lipid environments/conformations induced when clotting proteins bind to bilayers. Aim 4 will solve the structure of tissue factor on the membrane surface and identify conformational changes in tissue factor when it interacts with ligands. Together these studies will provide valuable new insights into the role of the membrane surface in blood clotting, at atomic-scale resolution. PUBLIC HEALTH RELEVANCE: Disorders of the blood clotting system represent the leading cause of disability and death in the United States, but we still have a very incomplete understanding of blood clotting reactions at the molecular level. These studies will shed new light on the regulation of the blood clotting system, with a particular focus on achieving a detailed understanding of how and why blood clotting reactions occur on membrane surfaces.