MOLECULAR MECHANISMS OF THROMBIN ACTION

凝血酶作用的分子机制

• 批准号: 3087750
• 负责人: DAVID HUNG
• 金额: $ 8.64万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3087750
• 关键词: X ray crystallography; Xenopus oocyte; biological signal transduction; blood vessel disorder; complementary DNA; coronary artery; drug design /synthesis /production; enzyme mechanism; immunoprecipitation; messenger RNA; molecular cloning; peptidases; platelet activation; protein sequence; protein structure function; proteolysis; receptor binding; site directed mutagenesis; thrombin

Coronary artery disease is the leading cause of morbidity and mortality the United States and Western Europe. Thrombin plays a critical role in the pathogenesis of coronary artery disease at many levels. Although it is perhaps best recognized as the serine protease responsible for the cleavage of fibrinogen to fibrin of fibrinogen to fibrin during blood clotting, thrombin also functions for a diverse number of cellular activities. It is the most potent physiological stimulus for platelet aggregation known and there are recent data to suggest that thrombin is a pivotal mediator of arterial thrombosis in vivo. Also well-documented are thrombin's mitogenicity in vascular smooth muscle cells, fibroblasts and splenocytes and chemotactic properties for monocytes. It therefore appears that in addition to its critical role in thrombosis, thrombin may play an important role in regulating the inflammatory and repair processes that accompany arterial wall injury. The long term goal of this project is to identify the molecules and molecular mechanisms that mediate thrombin activation of platelets and other cells. Attainment of this goal may facilitate the rational design of drugs capable of specifically antagonizing pathological thrombosis and cellular activation by thrombin. The specific aims of the work proposed herein are 1) to determine the importance of protease activity and specific thrombin structural determinants in cell and platelet activation, and other thrombin-specific functions, and 2) to obtain and characterize a cDNA clone for the thrombin receptor. The importance of thrombin's protease activity in signal transduction will be investigated using oligonucleotide-directed mutagenesis to create recombinant thrombin mutants that are catalytically inert but retain receptor binding ability. The structural domains that mediate thrombin signalling will be investigated using oligonucleotide-directed mutagenesis to delete or exchange those domains that differ from other serine proteases and therefore seem likely to be responsible for thrombin's unique spectrum of activity. The thrombin receptor will be isolated by the strategy of expression cloning in which microinjection of Xenopus oocytes with mRNA from thrombin-responsive cells confers thrombin responsiveness upon the oocytes. Alternative approaches to the isolation of the thrombin receptor are affinity purification of the receptor using a novel catalytically inert thrombin with unaltered binding affinity for its receptor and the cloning and expression of thrombin receptor candidates such as glycoprotein V.

冠状动脉疾病是发病率和死亡率的主要原因 美国和西欧。凝血酶在其中起着至关重要的作用 冠状动脉疾病的发病机制在多个层面上。虽然它是 也许最被认为是负责切割的丝氨酸蛋白酶 凝血过程中纤维蛋白原转化为纤维蛋白 纤维蛋白原转化为纤维蛋白， 凝血酶还对多种细胞活动起作用。这是 已知对血小板聚集最有效的生理刺激 最近的数据表明凝血酶是一个关键的介质 体内动脉血栓形成。凝血酶也有充分记录 血管平滑肌细胞、成纤维细胞和脾细胞的有丝分裂能力 和单核细胞的趋化特性。因此看来，在 除了在血栓形成中发挥关键作用外，凝血酶还可能发挥重要作用 在调节伴随的炎症和修复过程中的作用 动脉壁损伤。 该项目的长期目标是识别分子并 介导血小板凝血酶激活的分子机制 其他细胞。实现这一目标可能有助于合理设计 能够特异性拮抗病理性血栓形成的药物 凝血酶激活细胞。拟议工作的具体目标 这里是 1) 确定蛋白酶活性和特异性的重要性 细胞和血小板活化中的凝血酶结构决定因素，以及其他 凝血酶特异性功能，以及 2) 获得并表征 cDNA 克隆 对于凝血酶受体。 凝血酶的蛋白酶活性在信号转导中的重要性 使用寡核苷酸定向诱变进行研究以创建 重组凝血酶突变体具有催化惰性但保留 受体结合能力。介导凝血酶的结构域 将使用寡核苷酸定向诱变来研究信号传导 删除或交换那些与其他丝氨酸蛋白酶不同的域 因此似乎可能是凝血酶独特谱的原因 的活动。 凝血酶受体将通过以下策略分离 表达克隆，其中用 mRNA 显微注射非洲爪蟾卵母细胞 来自凝血酶反应性细胞的凝血酶反应性赋予 卵母细胞。 分离凝血酶受体的替代方法 使用新型催化惰性亲和纯化受体 对其受体和克隆具有未改变的结合亲和力的凝血酶 以及候选凝血酶受体（例如糖蛋白 V）的表达。