The Prothrombinase Complex: A Model of an Enzyme-Cofactor Complex

描述（由申请人提供）：止血是通过导致血栓形成（凝血）和溶解（纤溶）的两个级联的微妙平衡来维持的。凝血级联是通过形成三个连续的酶-辅因子复合物迅速导致大量凝血酶的产生而传播的。凝血酶原复合物由酶因子Xa和辅因子Va之间的非共价结合形成，是凝血酶的最终生成物。没有这种复杂的生理相关水平的凝血酶不能在出血发生前形成足够的时间形成血栓。虽然凝血酶原复合物的动力学已经非常详细，但导致酶活性大调节的Xa和Va因子之间的关联机制尚不清楚。分析酶-辅因子界面残基和复合物结构是了解其相互作用机理的必要条件。本提案的目的是：(1)确定辅助因子内部以及辅助因子Va和酶因子Xa之间非共价相互作用的分子机制，这些相互作用是形成完全活性的凝血酶原复合物所必需的；(2)测定凝血酶原复合物的x射线晶体结构。最近失活因子Va的晶体结构已经确定了辅助因子稳定的潜在区域，并导致几个计算机模型提出了酶-辅助因子界面上的相互作用区域。参与辅因子稳定和酶-辅因子相互作用的牵连残基突变将分析其对凝血酶原活性和调控的影响。酶和辅因子的协调突变将确定在界面上发现的非共价相互作用以及在凝血酶生成中的作用。凝血酶原复合物的x射线结构最有可能确定界面上的残基，以及识别额外的残基和相互作用。对所涉及的蛋白质和复合物的生化和结构的理解将使靶向药物设计能够帮助维持止血。超过40%的美国人患有某种形式的心血管疾病，因此对凝血级联进行药物干预和调节是一种临床需要。

DESCRIPTION (provided by applicant): Hemostasis is maintained through a delicate balance of two cascades leading to the formation (coagulation) and dissolution (fibrinolysis) of a clot. The coagulation cascade is propagated through formation of three successive enzyme-cofactor complexes that rapidly leads to generation of large quantities of thrombin. The prothrombinase complex, formed between a noncovalent association between the enzyme factor Xa and cofactor Va, serves as the final generator of thrombin. Without this complex physiologically relevant levels of thrombin cannot be formed in a sufficient amount of time to form a clot before hemorrhaging occurs. While the kinetics of the prothrombinase complex have been greatly detailed, the mechanism of association between factors Xa and Va that leads to large modulations in enzymatic activity is not. Analysis of the residues at the enzyme-cofactor interface and complex structure is necessary to understand the mechanism of interaction. The aims of this proposal are to: (1) determine the molecular mechanism of the noncovalent interactions, both within the cofactor as well as between the cofactor Va and enzyme factor Xa, required for formation of a fully active prothrombinase complex; and (2) determine the x-ray crystallographic structure of the prothrombinase complex. A recent crystal structure of inactivated factor Va has identified potential regions of cofactor stabilization and led to several computer models proposing regions of interaction at the enzyme-cofactor interface. Mutations of implicated residues involved in cofactor stabilization and enzyme- cofactor interactions will be analyzed for their effect on prothrombinase activity and regulation. Coordinated mutations on both enzyme and cofactor will identify the noncovalent interactions found at the interface and role in thrombin generation. The x-ray structure of the prothrombinase complex has the most potential for confirming residues at the interface as well as identifying additional residues and interactions. A biochemical and structural understanding of involved proteins and complexes will enable targeted drug design to assist in maintaining hemostasis. Pharmacological intervention and regulation of the coagulation cascade is a clinical necessity with more than 40% of the U.S. population suffering from some form of cardiovascular disease.