Structural Mechanisms for the Inhibition of Thrombosis

抑制血栓形成的结构机制

• 批准号: 6415060
• 负责人: JAMES Andrew HUNTINGTON
• 金额: $ 26.6万
• 依托单位: UNIVERSITY OF CAMBRIDGE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-21 至 2004-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6415060
• 关键词: X ray crystallography; activation product; antithrombins; carbohydrate structure; chemical kinetics; coagulation factor X; cofactor; conformation; enzyme activity; enzyme complex; enzyme inhibitors; enzyme mechanism; fibrin; gene mutation; heparin; ionic bond; mucopolysaccharides; protein binding; protein structure function; serine proteinases; structural biology; thrombin; thrombosis

DESCRIPTION (provided by applicant): The long-term objective of this research proposal is to understand the mechanisms by which the principal two plasma inhibitors of thrombin, antithrombin and heparin cofactor II, are activated by heparin-like glycosaminoglycans. Such an understanding will help explain dysfunction which leads to familial thrombophilia and aid in the development of novel therapeutic antithrombotic agents. This objective will largely be fulfilled through the successful completion of the specific aims which follow from the general hypotheses that: i) antithrombin and heparin cofactor II circulate in a kinetically inactive state; ii) binding to and activation by glycosaminoglycans involves a dramatic conformational change to a kinetically active state; iii) the shared serpin mechanismn of protease inhibition plays a critical role in facilitating release of the final complex from glycosaminoglycan and fibrin to allow for clearance and in situ degradation. The specific aims fall under three general questions: I) How does heparin accelerate antithrombin inhibition of thrombin? II) How is antithrombin activated towards factor Xa? and, III) How is heparin cofactor II activated towards thrombin? A combination of protein X-ray crystallographic and biochemical studies will be employed to answer these questions. The specific aims are: 1) To determine the structures of antithrombin and thrombin bound to heparin. 2) To determine the structure of the initial antithrombin-thrombin-heparin complex. 3) To determine the structure of the final antithrombin-thrombin complex. 4) To determine the structure of native and pentasaccharide-activated antithrombins. 5) To investigate the role of surface electrostatics in the allosteric activation of antithrombin. 6) To determine the structure of native and glycosaminoglycan bound heparin cofactor II. 7) To determine the structure of the initial heparin cofactor Il-thrombin complex. 8) To investigate the conformational link between glycosaminoglycan binding and release of the N-terminal tail. 9) To determine the conformational basis for cleavage of the N-terminal tail from the final heparin cofactor II-thrombin complex.

描述（由申请人提供）：本研究的长期目标 建议是了解机制，其中主要的两个等离子体 凝血酶、抗凝血酶和肝素辅因子II的抑制剂，被 肝素样糖胺聚糖。这样的理解将有助于解释 功能障碍，导致家族性血栓形成倾向，并有助于发展 新的治疗性抗血栓形成剂。这一目标在很大程度上将 通过成功完成以下具体目标来实现 根据以下一般假设：i）抗凝血酶和肝素辅因子II 以动力学非活性状态循环; ii）结合并活化 糖胺聚糖涉及到一个戏剧性的构象变化，以动力学 活性状态; iii）蛋白酶抑制的共享丝氨酸蛋白酶抑制剂机制起作用， 在促进最终复合物从 以允许清除和原位降解。 具体的目标属于三个一般问题：I）肝素如何 加速抗凝血酶抑制凝血酶？二、抗凝血酶是怎样的 对Xa因子有反应吗肝素辅因子II是如何被激活的 对凝血酶的反应结合蛋白质X射线晶体学和 生物化学研究将被用来回答这些问题。具体 目的是：1）确定抗凝血酶和凝血酶的结构， 肝素2)为了确定初始的结构， 抗凝血酶-凝血酶-肝素复合物3)为了确定 最终的抗凝血酶-凝血酶复合物。4)为了确定天然的 和戊糖醇激活的抗凝血酶。5)调查的作用 抗凝血酶变构活化中的表面静电。6)到 确定天然和糖胺聚糖结合的肝素辅因子的结构 二. 7)确定初始肝素辅因子II-凝血酶的结构 复杂. 8)探讨糖胺聚糖之间的构象联系 结合并释放N-末端尾。9)为了确定构象 从最终肝素辅因子裂解N-末端尾的基础 II-凝血酶复合物。