STRUCTURAL DETERMINANTS OF FACTOR XA FUNCTION

因子Xa是称为凝血酶原酶的酶复合物的丝氨酸蛋白酶组分，凝血酶原酶是唯一已知的凝血酶原生理激活剂。 这项工作的广泛的长期目标是确定FXa的结构决定因素，这些决定因素对其在凝血酶原酶复合物中的功能很重要。这项建议有两个具体目标。 在第一个目标中，我们将确定FXa的催化结构域中的表面暴露区域是否定义辅因子和底物外位点。 来自该目的的待测试的假设是，1）FXa上的162螺旋（残基162-170，胰凝乳蛋白酶编号）提供FVa的关键结合表面，和2）FXa上的带负电荷的39环（残基35-41）与其底物凝血酶原进行特异性静电接触。 在第二个目标中，我们将确定钠与FXa上的225环（残基220-226）的结合是否调节其活性。 我们的假设是，在缺乏钠结合的情况下，FXa识别大分子底物的能力被改变。 为了实现这些目标，我们将1）使用定点诱变制备FXa的重组变体，2）在哺乳动物表达系统中产生变体，3）表征蛋白质的以下方面：纯度、γ-羧基谷氨酸含量、凝血试验中的活性、显色底物和大分子底物的裂解以及与非蛋白和蛋白辅因子的相互作用。 突变的选择将基于FXa的可用晶体结构，以及先前定义同源酶复合物中分子决定簇的实验结果。 了解和识别凝血酶原酶的组装和功能中涉及的复杂分子相互作用是开发旨在控制凝血酶局部浓度的药理学药物的关键第一步。 此外，这一建议将有助于我们了解FXa的底物特异性的决定因素，这可能最终导致新的策略来调节这种关键的丝氨酸蛋白酶的催化特性。

Factor Xa is the serine protease component of the enzymatic complex termed prothrombinase, the only known physiological activator of prothrombin. The broad long-term objective of this work is to identify structural determinants on FXa that are important for its function within the prothrombinase complex. This proposal contains two specific aims. In the first aim, we will determine whether surface exposed regions in the catalytic domain of FXa define cofactor and substrate exosites. The hypotheses from this aim to be tested are, 1) the 162 helix on FXa (residues 162-170, chymotrypsin numbering) provides a critical binding surface for FVa, and 2) the negatively charged 39-loop on FXa (residues 35-41) makes specific electrostatic contacts with its substrate prothrombin. In the second aim, we will determine whether sodium binding to the 225-loop (residues 220-226) on FXa modulates its activity. It is our hypothesis that in the absence of sodium binding the ability of FXa to recognize macromolecular substrates is altered. To carry these aims out, we will 1) prepare recombinant variants of FXa using site-directed mutagenesis, 2) produce the variants in a mammalian expression system, and 3) characterize the proteins with respect to: purity, gamma-carboxyglutamic acid content, activity in clotting assays, cleavage of chromogenic and macromolecular substrates, and interactions with non-protein and protein cofactors. Choice of mutations will be based on the available crystal structure of FXa, and on previous experimental findings defining molecular determinants in homologous enzymatic complexes. Understanding and identifying the complex molecular interactions involved in the assembly and function of prothrombinase, is a key first step in the development of pharmacologic agents aimed at controlling local concentrations of thrombin. In addition, this proposal will contribute to our understanding of the determinants of FXa's substrate specificity, which may ultimately lead to new strategies for modulating the catalytic properties of this critical serine protease.