Function and dysfunction in human antithrombins

人类抗凝血酶的功能和功能障碍

• 批准号: 7166831
• 负责人: PETER G.W. GETTINS
• 金额: $ 36.58万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7166831
• 关键词: Antithrombins; Area; Binding Sites; Blood coagulation; Cardiovascular Diseases; Collaborations; Complex; Crystallography; Defect; Developed Countries; Development; Endopeptidases; Equilibrium; Factor Xa; Functional disorder; Genes; Goals; Heparin; Heparin Binding; Human; Inherited; Kinetics; Malignant Neoplasms; Mammalian Cell; Molecular; Mutation; Peptide Hydrolases; Predisposition; Process; Protease Inhibitor; Protein Family; Proteins; Recombinants; Robin bird; Role; Serine Proteinase Inhibitors; Serpins; Structure; Testing; Thermodynamics; Thrombin; Thrombosis; Variant; Venous Thrombosis; antithrombin III-protease complex; base; beta pleated sheet; glycosylation; inhibitor/antagonist; member

Inherited defects in single genes contribute significantly in many people to a predisposition to development of venous thrombosis, which in turn is a major contributor to the leading killer in industrialized countries, cardiovascular disease. One of the most important inherited defects is in the gene for antithrombin. Antithrombin is the principal inhibitor of the blood coagulation proteinases factor Xa and thrombin and is regulated by heparin. The long term goal of this proposal is to achieve an understanding of the molecular basis for defects in functioning of variant human antithrombins that result in thrombosis. This will be accomplished through elucidation first of the mechanisms of heparin activation and proteinase inhibition in normal antithrombin, and the ways in which mutations or changes in glycosylation alter either or both of these processes. The general hypotheses are (i) that the normal functioning of antithrombin can only be understood in terms of it being a serpin (member of the serine proteinase inhibitor superfamily) and of consequently being capable of undergoing necessary and dramatic conformational changes as part of both heparin binding and activation, and of proteinase inhibition and (ii) that, as a consequence of the need for antithrombin to fold as a metastable protein and to undergo conformational change as part of its function, it is prone to many more defects than other families of protein proteinase inhibitors which form simple lock-and-key type complexes. The specific areas are:- (1) To determine the gross structure of the thrombin-antithrombin complex. (2) To determine the conformational linkage between heparin binding and expulsion of residues of the reactive center of beta-sheet A. (3) To test whether the reactive center loop of antithrombin exists in an equilibrium between less reactive partially-inserted and more reactive fully loop expelled forms and that heparin activation results from a shift in this equilibrium. (4) To determine the role of basic residues in promoting the conformational change in the heparin binding site that results in expulsion of P15 and P14 residues of the reactive center loop. (5) To determine the basis for the dysfunction of naturally occurring human antithrombin variants. (6) To determine whether antithrombin is fucosylated in cancer and the functional consequences thereof. These specific aims will make extensive use of recombinant antithrombins expressed in mammalian cells that will be characterized by a combination of spectroscopic, thermodynamic and kinetic means. For antithrombins that have been activated by mutation, x-ray crystallography, through collaboration with Dr. Robin Carrell, will be used.

单个基因的遗传缺陷在许多疾病中起着重要作用。 人们容易患上静脉血栓， 反过来又是工业化国家中主要杀手的主要贡献者， 心血管疾病。 其中最重要的继承 抗凝血酶基因有缺陷 抗凝血酶是 凝血蛋白酶Xa因子和凝血酶抑制剂 并由肝素调节。 本提案的长期目标是 实现对缺陷的分子基础的理解， 导致血栓形成的变体人抗凝血酶的功能。 这将通过阐明第一机制来实现 肝素活化和蛋白酶抑制的作用， 以及糖基化的突变或变化改变 或者这两种过程。一般的假设是（i）， 抗凝血酶的正常功能只能从 它是丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制剂的成员 超家族）并且因此能够经历必要的 以及作为肝素结合和 激活，和蛋白酶抑制和（ii），作为结果， 需要抗凝血酶折叠为亚稳态蛋白， 经历构象变化作为其功能的一部分，它倾向于 比其他蛋白质蛋白酶抑制剂家族的缺陷更多 其形成简单的锁和钥匙类型的复合体。 具体领域包括： (1)测定凝血酶-抗凝血酶的宏观结构 复杂. (2)为了确定肝素与 β折叠反应中心残基的结合和排出 A. (3)检测抗凝血酶反应中心环是否存在 在较少反应性的部分插入和较多反应性的部分插入之间的平衡中， 反应性完全环排出形式，肝素活化导致 从这个平衡的转变中。 (4)确定基本的作用 促进肝素结合中构象变化的残基 位点，导致反应性蛋白的P15和P14残基排出。 中央回路。 (5)为了确定功能障碍的基础， 天然存在的人抗凝血酶变体。 (6)以确定 抗凝血酶是否在癌症中被岩藻糖基化， 其后果。 这些具体目标将广泛利用 在哺乳动物细胞中表达的重组抗凝血酶， 其特征在于光谱、热力学和 动力学手段 对于被突变激活的抗凝血酶， 通过与罗宾·卡雷尔博士的合作， 将用于

项目成果

Formation of the covalent serpin-proteinase complex involves translocation of the proteinase by more than 70 A and full insertion of the reactive center loop into beta-sheet A.

• DOI: 10.1073/pnas.96.9.4808
• 发表时间: 1999-04
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: E. Stratikos;P. Gettins

alpha1-Proteinase inhibitor forms initial non-covalent and final covalent complexes with elastase analogously to other serpin-proteinase pairs, suggesting a common mechanism of inhibition.

与其他丝氨酸蛋白酶抑制剂-蛋白酶对类似，α1-蛋白酶抑制剂与弹性蛋白酶形成初始非共价复合物和最终共价复合物，这表明存在共同的抑制机制。

• DOI: 10.1074/jbc.m311731200
• 发表时间: 2004
• 期刊: The Journal of biological chemistry
• 作者: Dobó,József;Gettins,PeterGW
• 通讯作者: Gettins,PeterGW

The allosteric mechanism of activation of antithrombin as an inhibitor of factor IXa and factor Xa: heparin-independent full activation through mutations adjacent to helix D.

作为因子 IXa 和因子 Xa 抑制剂的抗凝血酶激活的变构机制：通过与螺旋 D 相邻的突变实现不依赖肝素的完全激活。

• DOI: 10.1074/jbc.m113.510727
• 发表时间: 2013
• 期刊: The Journal of biological chemistry
• 作者: Dementiev,Alexey;Swanson,Richard;Roth,Ryan;Isetti,Giulia;Izaguirre,Gonzalo;Olson,StevenT;Gettins,PeterGW
• 通讯作者: Gettins,PeterGW