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DYSFUNCTION OF VARIANT HUMAN ANTITHROMBINS

人类抗凝血酶变体的功能障碍

基本信息

批准号：
2028808
负责人：
PETER G.W. GETTINS
金额：
$ 24.36万
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：
1993
资助国家：
美国
起止时间：
1993-01-01 至 1997-12-31
项目状态：
已结题

项目摘要

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.

工业化国家最常见的死亡原因是心血管疾病，其中血栓形成起着非常突出的作用。许多人容易发生血栓栓塞性疾病。并发症通过遗传缺陷的单一基因，其中其中最重要的是抗凝血酶III基因，因为抗凝血酶III作为一种蛋白酶抑制剂，血液凝固据估计，2%至5%的患者血栓栓塞性疾病的患者有这样的缺陷。本提案的长期目标是实现对人类抗凝血酶变异体功能缺陷的分子基础导致血栓形成，通过阐明肝素活化和蛋白酶抑制，以及测定突变改变其中一个或两个过程的方式。这种理解是合理设计治疗方案的前提干预措施。糖基化重组人的表达系统抗凝血酶将用于产生野生型和变体抗凝血酶。结构研究将不局限于一种技术，但将利用几种并行和互补的方法，包括NMR，CD， FTIR和荧光光谱。有待实现的具体目标在建议的资助期内， (i)天然抗凝血酶中肝素结合位点的表征 III，其中构成肝素结合位点或受肝素结合干扰的细胞。 (ii)反应中心区结构的确定天然抗凝血酶III。这将测试目前的模式，该区域在天然状态下是α-螺旋是正确的，确定蛋白酶扰动后的后续变化，或肝素 (iii)确定传递至细胞的构象变化反应性中心区域随之肝素结合，因为这种变化是抗凝血酶激活的必要组成部分。 (iv)正常和自然发生的病理变异的比较抗凝血酶III 通过这种方式，的变体抗凝血酶将被揭示，然后可以关联功能障碍的表现。 (v)适于衍射的天然抗凝血酶晶体的生长问题研究