FUNCTION AND DYSFUNCTION IN HUMAN ANTITHROMBINS

单基因的遗传缺陷在许多疾病中起重要作用 人们对静脉血栓的易感性，这 反过来又是工业化国家头号杀手的主要贡献者 国家，心血管疾病。最重要的继承者之一 缺陷存在于抗凝血酶基因中。抗凝血酶是主要的 凝血酶抑制因子Xa和凝血酶 并受肝素调节。这项提议的长期目标是 实现对缺陷的分子基础的理解 导致血栓形成的变种人类抗凝血酶的功能。 这将通过首先阐明机制来实现。 在正常的抗凝血酶中肝素的激活和蛋白酶的抑制， 以及糖基化的突变或变化改变 或者这两个过程。一般假设是：(I) 抗凝血酶的正常功能只能从以下方面来理解 它是一种丝氨酸(丝氨酸蛋白酶抑制剂的成员 超级家庭)，并因此能够经历必要的 和显著的构象变化作为肝素结合和 激活和抑制蛋白酶，以及(Ii)其结果是 需要抗凝血酶以亚稳态蛋白的形式折叠并 作为其功能的一部分，它容易发生构象变化 与其他蛋白酶抑制剂家族相比，缺陷多得多 它们形成简单的锁和钥匙类型的复合体。具体范围如下： (1)测定凝血酶-抗凝血酶的大体结构 很复杂。(2)确定肝素之间的构象连接 β-折叠反应中心残基的结合和排出 A.(3)检测抗凝血酶反应中心环是否存在 在活性较低的部分插入和更多活性之间的平衡 反应性全环排出形式和肝素激活结果 从这种平衡的转变中。(四)确定基层组织的作用 促进肝素结合构象变化的残基 导致活性物质的P15和P14残基排出的部位 中心环路。(5)确定功能障碍的基础 自然产生的人类抗凝血酶变种。(六)确定 抗凝血酶是否在癌症和功能性疾病中被岩藻糖基化 由此产生的后果。这些具体目标将广泛使用 在哺乳动物细胞中表达的重组抗凝血酶将被 以光谱、热力学和化学的结合为特征 动力学手段。对于被突变激活的抗凝血酶， X射线结晶学，通过与罗宾·卡雷尔博士合作， 将会被使用。

Inherited defects in single genes contribute significantly in many people to a predisposition to development of venous thrombosis, which in turn is a major contributor to the leading killer in industrialized countries, cardiovascular disease. One of the most important inherited defects is in the gene for antithrombin. Antithrombin is the principal inhibitor of the blood coagulation proteinases factor Xa and thrombin and is regulated by heparin. 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. This will be accomplished through elucidation first of the mechanisms of heparin activation and proteinase inhibition in normal antithrombin, and the ways in which mutations or changes in glycosylation alter either or both of these processes. The general hypotheses are (i) that the normal functioning of antithrombin can only be understood in terms of it being a serpin (member of the serine proteinase inhibitor superfamily) and of consequently being capable of undergoing necessary and dramatic conformational changes as part of both heparin binding and activation, and of proteinase inhibition and (ii) that, as a consequence of the need for antithrombin to fold as a metastable protein and to undergo conformational change as part of its function, it is prone to many more defects than other families of protein proteinase inhibitors which form simple lock-and-key type complexes. The specific areas are:- (1) To determine the gross structure of the thrombin-antithrombin complex. (2) To determine the conformational linkage between heparin binding and expulsion of residues of the reactive center of beta-sheet A. (3) To test whether the reactive center loop of antithrombin exists in an equilibrium between less reactive partially-inserted and more reactive fully loop expelled forms and that heparin activation results from a shift in this equilibrium. (4) To determine the role of basic residues in promoting the conformational change in the heparin binding site that results in expulsion of P15 and P14 residues of the reactive center loop. (5) To determine the basis for the dysfunction of naturally occurring human antithrombin variants. (6) To determine whether antithrombin is fucosylated in cancer and the functional consequences thereof. These specific aims will make extensive use of recombinant antithrombins expressed in mammalian cells that will be characterized by a combination of spectroscopic, thermodynamic and kinetic means. For antithrombins that have been activated by mutation, x-ray crystallography, through collaboration with Dr. Robin Carrell, will be used.