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Molecular Basis of Blood Coagulation Regulation

凝血调节的分子基础

基本信息

批准号：
9438409
负责人：
Steven T. Olson
金额：
$ 39.18万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-03-01 至 2020-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9438409
关键词：
Affinity Amino Acids Anticoagulants Antithrombins Bacteria Binding Binding Sites Biochemical Biological Assay Biotechnology Blocking Antibodies Blood Coagulation Disorders Blood coagulation Burial Cardiovascular Diseases Cause of Death Charge Coagulation Process Collaborations Communication Complex Coupling Disease Disulfides Down-Regulation Embryo Engineering Epitopes Equilibrium Factor IXa Factor Xa Fluorescence Generations Genes Hemophilia A Hemorrhage Hemostatic function Heparin Heparin Binding Human Hydrophobicity In Vitro Instruction Isotope Labeling Kinetics Knockout Mice Label Mediating Membrane Modeling Molecular Molecular Conformation Mus Mutagenesis Mutate Mutation Myocardial Infarction N-terminal Oxides Patients Peptide Hydrolases Pharmacologic Substance Phenotype Physiological Plasma Protease Inhibitor Protein Family Proteins Regulation Reporter Reporting Risk Factors Role Scanning Serpin Superfamily Serpins Signal Transduction Site Site-Directed Mutagenesis Sodium Chloride Stroke Tail Testing Thermodynamics Thrombin Thromboplastin Time Universities Up-Regulation Variant base biophysical techniques cancer procoagulant cofactor experimental study factor V Leiden fluorophore gain of function high throughput screening human subject instrument knockout animal novel strategies novel therapeutics oxidation plasma protein Z scaffold small molecule small molecule libraries transmission process virtual

项目摘要

Serpin family protein protease inhibitors function as key anticoagulant regulators of blood coagulation proteases. Two serpins, antithrombin and protein Z-dependent protease inhibitor (ZPI), are known to inhibit procoagulant proteases in a manner that is regulated by cofactors and dependent on the functional state of the proteases, but the molecular details of this complex regulatory mechanism are poorly understood. The physiologic importance of these serpins in regulating coagulation proteases is borne out by the observations that knocking out the mouse genes results in embryonic lethality due to a consumptive coagulopathy in the case of antithrombin and a prothrombotic phenotype when mice are bred on a factor V Leiden background in the case of ZPI. Our proposed studies seek to build on our prior studies to advance molecular understanding of the cofactor-dependent regulation of blood coagulation proteases by these serpins. With respect to antithrombin, our recent studies have suggested an important revision of the allosteric mechanism of activation of this serpin by heparin in showing that activation is mediated principally by the mitigation of repulsive interactions with factor Xa and factor IXa within an antithrombin exosite rather than by provision of an attractive exosite. Our proposed continuation studies seek to characterize the molecular determinants in antithrombin of protease binding exosites and of the allosteric communication network involved in transmitting activating conformational changes from the heparin binding site to the protease binding site. With respect to ZPI, our studies seek to elucidate the physiologic complexes of factor Xa that are targeted by ZPI and its cofactor, protein Z, establish the role of the unique N-terminal tail of ZPI in anticoagulant regulation of factor Xa and discover small molecules that disrupt the ZPI-protein Z complex and downregulate the ZPI anticoagulant mechanism as a potential novel therapy for restoring hemostasis in hemophilia bleeding disorders. Gain of function studies in which the serpin scaffold of α1-protease inhibitor is used to graft the molecular determinants of the protein Z binding site and protease binding sites onto ZPI will provide a stringent test of the minimal determinants of cofactor and protease recognition by this serpin.

丝氨酸蛋白酶抑制剂家族蛋白蛋白酶抑制剂是凝血蛋白酶的关键抗凝调节剂。已知两种丝氨酸蛋白酶抑制剂（抗凝血酶和蛋白 Z 依赖性蛋白酶抑制剂 (ZPI)）能够以辅因子调节并依赖于蛋白酶功能状态的方式抑制促凝血蛋白酶，但人们对这种复杂调节机制的分子细节知之甚少。这些丝氨酸蛋白酶抑制剂在调节凝血蛋白酶方面的生理学重要性得到了观察结果的证实，即敲除小鼠基因会导致胚胎死亡，因为在抗凝血酶的情况下会出现消耗性凝血病，而在ZPI的情况下，当小鼠在莱顿因子V背景下饲养时会出现促血栓表型。我们提出的研究旨在以我们之前的研究为基础，以促进对这些丝氨酸蛋白酶抑制剂对凝血蛋白酶的辅因子依赖性调节的分子理解。关于抗凝血酶，我们最近的研究建议对肝素激活丝氨酸蛋白酶抑制剂的变构机制进行重要修改，表明激活主要是通过减轻抗凝血酶外部位点内与因子Xa和因子IXa的排斥相互作用来介导的，而不是通过提供有吸引力的外部位点来介导的。我们提出的继续研究旨在表征蛋白酶结合外位点的抗凝血酶和参与将激活构象变化从肝素结合位点传递到蛋白酶结合位点的变构通讯网络的分子决定因素。关于 ZPI，我们的研究旨在阐明 ZPI 及其辅因子蛋白 Z 所靶向的因子 Xa 的生理复合物，建立 ZPI 独特的 N 末端尾部在因子 Xa 抗凝调节中的作用，并发现破坏 ZPI-蛋白 Z 复合物并下调 ZPI 抗凝机制的小分子，作为恢复止血的潜在新疗法血友病出血性疾病。使用 α1-蛋白酶抑制剂的丝氨酸蛋白酶抑制剂支架将蛋白质 Z 结合位点和蛋白酶结合位点的分子决定因素移植到 ZPI 上的功能获得研究将提供对该丝氨酸蛋白酶抑制剂识别辅因子和蛋白酶的最小决定因素的严格测试。