MOLECULAR BASIS OF BLOOD COAGULATION REGULATION

这个项目的总体目标是阐明分子 丝氨酸对凝血酶的调节机制 抑制物，抗凝血酶(AT)；即是什么支配AT的特异性 它的目标蛋白水解酶，AT是通过什么机制捕获这些 稳定复合体中的酶，肝素是如何提高这一速度的 陷阱和其他调节因子在哪些方面起到保护蛋白酶的作用 它们的活动地点，并在它们逃脱时促进它们的失活 从这些网站？我们将确定AT对其特异性是否 靶标蛋白水解酶是由暴露的 酶活性部位区域的AT反应位点环和 肝素是否通过增强这种互补作用来激活AT 用的是蛋白酶。AT捕获酶的详细机制 稳定的络合物将被进一步阐明。美国政府的角色 构象变化，其中AT的暴露的反应部位环是 作为中心链插入蛋白质核心的Aβ-折叠， 将在这次诱捕中进行评估。我们将确定这是否 AT类底物相互作用引起的构象变化 对于这种酶，构象变化是否会将酶困在 这种底物反应的中间阶段以及是否有一些 酶可以通过完成反应的裂解来逃脱陷阱 在陷阱发生之前绑定。我们将进一步阐明其作用机制。 AT的肝素激活。AT Will的特异性肝素结合区 是本地化的，并且互动负责引发 AT的构象变化将被描绘出来。我们将决定 肝素诱导的构象变化是否导致 AT与某些蛋白水解酶的反应性增强及其是否起作用 只是为了促进AT与肝素对其他蛋白酶的紧密结合 反应，与蛋白水解酶的额外结合肝素是 负责增强AT与这些后一种酶的反应性。 我们最终将评估辅因子蛋白和/或酶原结构域 酶激活时释放的物质促进了蛋白酶的失活 AT和AT-肝素以及这些酶是否与 磷脂表面拮抗这种促进作用。这些研究将 利用经典的热力学和动力学方法来评估 AT、蛋白水解酶、肝素与其他物质的相互作用和反应 用于定位重组AT的效应子和定点突变 调节这些相互作用和反应的AT的功能位点。 预计这些研究将提供有关结构的新信息。 抗凝作用的基础及抗凝的一般机制 丝氨酸抑制剂的作用。对调节因素的阐明 AT-蛋白酶反应应该进一步提供一个更好的理解 凝血局部化机制及血管内皮生长因子的表达 凝血酶活性。最后，一个理性的基础， 含改良抗凝剂的重组ATS或肝素的设计 抗凝治疗的疗效是可以预期的。

The overall objective of this project is to elucidate the molecular mechanisms of regulation of blood coagulation proteinases by the serpin inhibitor, antithrombin (AT); i.e. what governs the specificity of AT for its target proteinases, what is the mechanism by which AT traps these enzymes in stable complexes, how does heparin enhance the rate of this trapping and what other modulating factors act to protect proteinases at their site of action and to promote their inactivation when they escape from these sites? We will determine whether the specificity of AT for its target proteinases is dictated by the complementarity of an exposed reactive-site loop of AT for the active-site region of the enzyme and whether heparin activates AT by enhancing this complementary interaction with proteinases. The detailed mechanism of AT trapping of enzymes in stable complexes will further be elucidated. The role of the conformational change, in which the exposed reactive-site loop of AT is inserted as a central strand into the A beta-sheet of the protein core, in this trapping will be assessed. We will determine whether this conformational change is induced by a substrate-like interaction of AT with the enzyme, whether the conformational change traps the enzyme at an inter-mediate stage of this substrate reaction and whether some of the enzyme can escape the trapping by completing the cleavage of the reactive bond before trapping occurs. We will further elucidate the mechanism of heparin activation of AT. The specific heparin binding region of AT will be localized and the interactions responsible for inducing a conformational change in AT will be delineated. We will determine whether the heparin-induced conformational change is responsible for the enhanced reactivity of AT with certain proteinases and whether it acts only to promote tight binding of AT to heparin for other proteinase reactions, with the additional binding of the proteinase to heparin being responsible for the enhanced reactivity of AT with these latter enzymes. We will finally assess whether cofactor proteins and/or proenzyme domains released upon enzyme activation promote the inactivation of proteinases by AT and AT-heparin and whether the binding of these proteinases to phospholipid surfaces antagonizes this promotion. These studies will utilize classical thermodynamic and kinetic approaches for evaluating the interactions and reactions between AT, proteinases, heparin and other effectors and site-directed mutagenesis of recombinant AT for mapping the functional sites of AT which mediate these interactions and reactions. The studies are expected to provide new information on the structural basis of AT anticoagulant action as well as the general mechanism of action of serpin inhibitors. Elucidation of the factors which modulate AT-proteinase reactions should further provide an increased understanding of the mechanisms of localization of clotting and the expression of the activity of clotting proteinases. Finally, a rational basis for the design of recombinant ATs or of heparins with improved anticoagulant efficacy for anticoagulant therapy is anticipated.