Molecular Basis of Blood Coagulation Regulation

凝血调节的分子基础

• 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
• 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

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.

丝氨酸蛋白水解酶抑制剂是凝血蛋白水解酶的关键抗凝调节剂。抗凝血酶和蛋白Z依赖的蛋白水解酶抑制物(ZPI)是两种已知的抑制促凝血酶的蛋白，其抑制方式受辅因子的调节，并且依赖于蛋白水解酶的功能状态，但这种复杂的调节机制的分子细节尚不清楚。这些蛇类在调节凝血酶方面的生理重要性得到了以下观察的证实：敲除小鼠基因会导致胚胎死亡，这是由于在抗凝血酶的情况下发生消耗性凝血障碍，而在ZPI的情况下，小鼠在因子V莱顿背景下繁殖时会出现血栓前表型。我们提出的研究试图建立在我们先前研究的基础上，以促进对这些蛇类对凝血酶的辅因子依赖的调节的分子理解。在抗凝血酶方面，我们最近的研究对肝素激活该蛇纹素的变构机制进行了重要的修订，表明激活主要是通过减轻与抗凝血酶外位内的Xa因子和IXa因子的排斥相互作用而不是通过提供有吸引力的外位而实现的。我们的后续研究试图确定抗凝血酶的分子决定因素，包括酶结合外切体和变构通讯网络，该网络参与将激活的构象变化从肝素结合部位传递到蛋白酶结合部位。关于ZPI，我们的研究试图阐明以ZPI及其辅因子蛋白Z为靶点的Xa因子的生理复合体，确定ZPI独特的N端尾巴在Xa因子抗凝调节中的作用，并发现破坏ZPI-蛋白Z复合体并下调ZPI抗凝机制的小分子，作为一种潜在的新疗法来恢复血友病出血的止血。在功能研究中，使用α1-蛋白酶抑制剂的丝氨酸支架将蛋白Z结合位点和蛋白酶结合位点的分子决定因素嫁接到ZPI上，这将为该丝氨酸识别辅因子和蛋白酶的最小决定因素提供严格的测试。