Protein S Anticoagulant Activity: Biochemical Mechanisms and Structural Studies

 描述（由申请人提供）：项目摘要/摘要血栓形成是美国的主要死亡原因。它是由凝血因子凝血酶的过度产生引起的。在正常止血过程中，凝血酶的产生受到几种抗凝蛋白的严格调节，包括蛋白S（PS）。PS是一种重要的抗凝蛋白，在人类中完全缺乏，在出生时产生严重的，危及生命的血栓形成。然而，PS介导其抗凝活性的生化机制知之甚少。PS是另外两种内源性抗凝剂组织因子途径抑制剂α（TFPIa）和活化蛋白C（APC）的辅因子。PS/TFPIa抑制凝血因子Xa（FXa），而PS/APC蛋白水解灭活因子Va（FVa）。FVa和FXa联合收割机形成凝血酶原酶，这是一种将凝血酶原转化为凝血酶的强大酶复合物。凝血酶原酶在血液凝固的传播阶段对PS/APC或PS/TFPIa的失活具有抗性，但其活性受到明显调节，因为在正常止血期间血栓不会扩散到整个脉管系统。PS促进凝血酶原酶的两种蛋白组分FVa（作为APC辅因子）和FXa（作为TFPIa辅因子）的失活，表明其可调节凝血酶原酶活性。我们假设PS/TFPIa和PS/APC抗凝系统的协调作用将产生凝血酶原酶的有效抑制。这一假设将通过补充的生化和结构研究进行检验。K99将专注于使用我在博士后研究期间建立的测定进行生化研究。他们将进一步定义PS增强TFPIa抑制活性的机制（具体目标1.1），并确定PS/TFPIa对FXa的抑制如何改变PS/APC蛋白水解FVa的能力，反之亦然（具体目标1.2）。R 00将通过评估不同生理膜表面对凝血酶生成调节的贡献来进一步扩展这项工作（具体目标1.3）。这些功能研究将与结构研究相关联。K99将专注于开发PS/TFPIa和PS/APC复合物的分子模型（具体目标2.1）。R 00将通过单独和复合地产生PS、TFPIa和APC的各个结构域的晶体结构来扩展这项工作（具体目标2.2和2.3）。这些研究将与职业发展培训相吻合，其中包括通过课程和Stephen Everse博士实验室工作的结合，重点关注结构生物学培训。这将使我的工作区别于我的导师，并使我能够建立一个独立的研究生涯，研究止血蛋白的生物化学。

 DESCRIPTION (provided by applicant): Project Summary/Abstract Thrombosis is a leading cause of death in the United States. It is caused by excessive production of the blood clotting factor thrombin. During normal hemostasis, thrombin generation is tightly regulated by several anticoagulant proteins, including protein S (PS). PS is an important anticoagulant protein in humans with total deficiency producing severe, life-threatening thrombosis at birth. However, the biochemical mechanism(s) by which PS mediates its anticoagulant activity are poorly understood. PS is a cofactor for two other endogenous anticoagulants, tissue factor pathway inhibitor alpha (TFPIa) and activated protein C (APC). PS/TFPIa inhibits coagulation factor Xa (FXa), while PS/APC proteolytically inactivates factor Va (FVa). FVa and FXa combine to form prothrombinase, the powerful enzymatic complex that converts prothrombin to thrombin. Prothrombinase is resistant to inactivation by either PS/APC or PS/TFPIa during the propagation phase of blood clotting, yet its activity is clearly regulated because thrombi do not spread throughout the entire vasculature during normal hemostasis. PS promotes the inactivation of both protein components of prothrombinase, FVa (as an APC cofactor) and FXa (as a TFPIa cofactor), suggesting that it may modulate prothrombinase activity. We hypothesize that coordinated action of the PS/TFPIa and PS/APC anticoagulant systems will produce efficient inhibition of prothrombinase. This hypothesis will be tested via complimentary biochemical and structural studies. The K99 will focus on biochemical studies using assays established during my post-doctoral research. They will further define the mechanism by which PS enhances TFPIa inhibitory activity (Specific Aim 1.1) and determine how FXa inhibition by PS/TFPIa alters the ability of PS/APC to proteolytically inactivate FVa and vice versa (Specific Aim 1.2). The R00 will extend this work further by assessing the contribution of different physiologic membrane surfaces to the regulation of thrombin generation (Specific Aim 1.3). These functional studies will be correlated with structural studies. The K99 will focus on development of molecular models of the PS/TFPIa and PS/APC complexes (Specific Aim 2.1). The R00 will extend this work by producing crystal structures of individual domains of PS, TFPIa, and APC, separately and in complex (Specific Aims 2.2 and 2.3). These studies will coincide with career development training, which will include a focus on training in structural biology, through a combination of courses and work in the laboratory of Dr. Stephen Everse. This will differentiate my work from that of my mentor and enable me to establish an independent research career studying the biochemistry of hemostatic proteins.