STRUCTURAL DETERMINANTS OF FACTOR XA FUNCTION

凝血因子Xa是被称为凝血酶原酶的酶复合体中的丝氨酸蛋白酶成分，是唯一已知的凝血酶原的生理激活剂。这项工作的长期目标是确定FXA上的结构决定因素，这些决定因素对FXA在凝血酶原酶复合体中的功能至关重要。这项提议包含两个具体目标。在第一个目标中，我们将确定FXA催化结构域中的表面暴露区域是否定义辅因子和底物外露。基于这一目标要检验的假设是，1)FXA上的162个螺旋(残基162-170，糜蛋白酶编号)为FVA提供了一个关键的结合表面，以及2)FXA上带负电荷的39环(残基35-41)与其底物凝血酶原发生特异的静电接触。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，在没有钠结合的情况下，FXA识别大分子底物的能力会改变。为了实现这些目标，我们将1)利用定点突变技术制备FXA的重组变异体，2)在哺乳动物表达系统中产生变异体，3)从纯度、γ-羧基谷氨酸含量、凝血活性、发色底物和大分子底物的裂解以及与非蛋白质和蛋白质辅助因子的相互作用等方面对蛋白质进行鉴定。突变的选择将基于FXA的可用晶体结构，以及之前定义同源酶复合体中分子决定因素的实验结果。了解和确定凝血酶原酶的组装和功能所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物的关键第一步。此外，这一建议将有助于我们理解FXA底物专一性的决定因素，这最终可能导致调节这种关键丝氨酸蛋白酶的催化性质的新策略。

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.