PROTEASE SPECIFICITY IN THE CONTROL OF HEMOSTASIS

控制止血的蛋白酶特异性

基本信息

批准号：
3366674
负责人：
WHYTE G OWEN
金额：
$ 31.76万
依托单位：
MAYO CLINIC ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-02-01 至 1997-01-31
项目状态：
已结题

项目摘要

The hemostasis system is subjected to exacting regulation to meet on one hand the demands of homeostasis while on the other to avert decompensation towards thrombosis which, in context of arterial disease, is the leading cause of morbidity and mortality in western cultures. The proposed research is aimed broadly at mechanisms that limit the activity of the hemostasis system to sites of injury, with a view of the long term goal of enabling strategies for detection, suppression and ultimately prevention of thrombosis. The focus of the proposal is on mechanisms of regulation by and of the proteases that comprise the hemostasis system, particularly the control of protease specificity by regulatory subunits, by substrate structure and by inhibitors. The focal protease is thrombin, which has a plastic specificity determined by a variety of plasma and cellular effectors including heparin and other polyanions, acidic peptides, a regulatory subunit (thrombomodulin) and inhibitors. A major aim of the proposal elucidation of tertiary structural details on thrombin that are involved in interactions with effectors. The major experimental approaches is measurement of the influence of effector binding on the rate constants of reductive methylation of every lysyl residue in thrombin. In parallel, the capacity of each effector to compete with each other will be evaluated. The goal is a tertiary/quaternary structure for each thrombin-effector dimer. A related but experimentally distinct aim is the nature of the platelet membrane protein that interacts with thrombin to initiate platelet activation. The working hypothesis is that hydrolysis of a membrane protein is the initiating event. The protein will be characterized by the relative susceptibility of platelet activation to thrombins or thrombin derivatives having a broad and non-parallel distribution of specific activities toward known thrombin substrates. The thrombins will be purified from plasmas of mammals, birds, reptiles and fish, and a limited group of chemical and genetic mutants will be used. The goal is characterization of the protein sufficiently to enable its physical identification and isolation. Many of the technical developments will be capitalized on to develop a thorough, conventional enzymology of the cognate protease, factor IXa, which is likewise regulated but is poorly characterized as an enzyme. The goal is an understanding of factor IXa at a level comparable to that of thrombin. Included in the goal is the regulation of factor IXa and its substrate/product factor X/Xa by the microvascular endothelium. The latter goal will be addressed in perfused rat hearts.

止血系统受到严格的调节，以达到一个将体内平衡的需求交给另一个，以避免代偿失调迈向血栓形成，在动脉疾病的背景下是领先的西方文化中发病和死亡率的原因。提议研究广泛针对限制活性的机制止血系统到受伤部位，以期为长期目标实现检测，抑制和最终预防的策略血栓形成。该提案的重点是通过以及构成止血系统的蛋白酶，特别是通过底物控制调节亚基的蛋白酶特异性结构和抑制剂。焦点蛋白酶是凝血酶，它具有由多种血浆和细胞确定的塑性特异性效应子，包括肝素和其他聚元，酸性肽，A 调节亚基（血栓形成蛋白）和抑制剂。一个主要目的提议阐明凝血酶的三级结构细节参与与效应子的互动。主要的实验方法是测量效应子结合对速率常数的影响凝血酶中每个赖氨酸残基的还原性甲基化。并联，将评估每个效应器与彼此竞争的能力。目标是每个凝血酶效应器的第三/第四纪结构二聚体。相关但实验上独特的目标是与凝血酶相互作用以启动血小板的血小板膜蛋白激活。工作假设是膜的水解蛋白质是起始事件。该蛋白质的特征是血小板激活对凝血酶或凝血酶的相对敏感性特异性的衍生物具有广泛且非并行分布针对已知凝血酶底物的活动。血栓将是从哺乳动物，鸟类，爬行动物和鱼的等离子体中纯化，有限将使用一组化学和遗传突变体。目标是足够的蛋白质表征以使其物理识别和隔离。许多技术发展将是大写以开发出一种彻底的常规酶学同源蛋白酶，因子IXA，同样受调节，但很差以酶为特征。目标是对IXA因子的理解一个与凝血酶相当的水平。目标包括对因子IXA及其底物/产品因子X/XA的调节微血管内皮。后一个目标将以灌注来解决老鼠心。