BASIS FOR DYSFUNCTION OF VARIANT HUMAN ANTITHROMBINS

变体人抗凝血酶功能障碍的基础

• 批准号: 3368387
• 负责人: PETER G.W. GETTINS
• 金额: $ 21.36万
• 依托单位: VANDERBILT UNIVERSITY
• 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 1993-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3368387
• 关键词: X ray crystallography; antithrombin III; binding proteins; blood coagulation; coagulation factor X; conformation; crystallization; electron spin resonance spectroscopy; fluorescence spectrometry; gene mutation; glycoprotein biosynthesis; heparin; human tissue; infrared spectrometry; nuclear magnetic resonance spectroscopy; protease inhibitor; protein structure function; recombinant proteins; site directed mutagenesis; thrombin; thrombosis

The single most common cause of death in industrialized countries is cardiovascular disease, in which thrombosis plays a very prominent role. Many people are predisposed to development of thromboembolic complications through inheritance of defects in single genes, of which one of the most important is the gene for antithrombin III, since antithrombin III plays a critical role as an inhibitor of proteinases of blood coagulation. It has been estimated that from 2 to 5% of patients with thromboembolic disorders have such a deficiency. 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, through elucidation of the mechanisms of heparin activation and proteinase inhibition, and the determination of the ways in which mutations alter either or both of these processes. Such understanding is a prerequisite for rational design of therapeutic interventions. An expression system for glycosylated recombinant human antithrombin will be used to produce wild type and variant antithrombins. Structural studies will not be confined to one technique, but will utilize several parallel and complementary approaches including NMR, CD, FTIR, and fluorescence spectroscopies. Specific goals to be accomplished within the proposed grant period are: (i) Characterization of the heparin binding site in native antithrombin III, in which residues that constitute the heparin binding site(s) or are perturbed by heparin binding will be identified. (ii) Determination of the structure of the reactive center region of native antithrombin III. This will test whether the current model that this region is alpha-helical in the native state is correct and permit determination of subsequent changes upon perturbation by proteinases or heparin. (iii) Determination of the conformational changes transmitted to the reactive center region consequent to heparin binding, since such changes are a necessary part of antithrombin activation. (iv) Comparison of normal and naturally occurring pathological variants of antithrombin III. In this way alterations in structure and mechanism of the variant antithrombins will be revealed and can then be correlated with the manifestations of the dysfunction. (v) Growth of crystals of native antithrombin suitable for diffraction studies.

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