DYSFUNCTION OF VARIANT HUMAN ANTITHROMBINS

在工业化国家，最常见的单一死因是 心血管疾病，其中血栓形成起着非常突出的作用。 许多人易患血栓栓塞症 通过单基因缺陷的遗传导致的并发症，其中 其中最重要的是抗凝血酶III的基因，因为 抗凝血酶III作为一种蛋白水解酶的抑制物起着至关重要的作用 血液凝结。据估计，2%到5%的患者 患有血栓栓塞症的人就有这样的缺乏症。 这项建议的长期目标是实现对 变异型人抗凝血酶功能缺陷的分子基础 通过阐明血管紧张素转换酶的作用机制 肝素活化和蛋白水解酶抑制，并测定 突变改变这两个过程中的一个或两个的方式。 这样的理解是合理设计治疗性药物的前提 干预措施。一种糖基化重组人的表达系统 抗凝血酶将用于生产野生型和变异型抗凝血酶。 结构研究不会局限于一种技术，而是 利用几种并行和互补的方法，包括核磁共振、CD、 傅立叶变换红外光谱和荧光光谱。要实现的具体目标 在建议的授权期内，下列项目包括： (I)天然抗凝血酶中肝素结合部位的鉴定 III，其中构成肝素结合位点的残基(S)或 被肝素结合所扰乱的将被识别。 (Ii)活性中心区域结构的测定 天然抗凝血酶III。这将测试目前的模型是否 这个区域在原生状态下是α螺旋的，是正确的和允许的 蛋白水解酶或蛋白水解酶干扰后后续变化的测定 肝素。 (3)确定传递给 肝素结合导致的反应中心区域，因为这样的变化 是抗凝血酶激活的必要部分。 (四)比较正常和自然发生的病理变异 抗凝血酶III。通过这种方式结构和机制的改变 的变种抗凝血酶将被揭示，然后可以相互关联 伴随着功能障碍的表现。 (V)适合于衍射的天然抗凝血酶晶体的生长 学习。

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.