Macromolecular Substrate Recognition in Blood Coagulation

凝血中的大分子底物识别

• 批准号: 7663365
• 负责人: Sriram Krishnaswamy
• 金额: $ 49.03万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7663365
• 关键词: Active Sites; Behavior; Binding; Biological; Biology; Blood; Blood Clot; Blood Platelets; Blood coagulation; CD46 Antigen; Cell surface; Chemistry; Cleaved cell; Coagulation Process; Docking; Endothelial Cells; Enzymatic Biochemistry; Enzyme Precursors; Enzymes; Exhibits; Factor Va; Fluorescence Resonance Energy Transfer; Goals; Hemostatic Agents; Hemostatic function; Individual; Kinetics; Lead; Life; Membrane; Multienzyme Complexes; Pathway interactions; Physiological; Play; Proteins; Prothrombin; Reaction; Relaxation; Research; Resolution; Role; Series; Serine Protease; Site; Specificity; Substrate Interaction; Substrate Specificity; Testing; Thermodynamics; Thromboplastin; aspergillopepsin II; base; carboxylation; catalyst; cell type; cofactor; conformational conversion; design; human disease; insight; meizothrombin; novel; novel strategies; proteinase In; response

The specific proteolytic activation steps of blood coagulation are catalyzed by serine proteinases that are homologous to each other and to the archetypal serine proteinases <of digestion. We will use prothrombin activation catalyzed by the prothrombinase complex as a paradigm for specific macromolecular substrate recognition and cleavage by coagulation complexes to investigate the basis for enzymic function. Our studies are focused on the central role of extended interactions between substrate and enzyme in driving the action of prothrombinase on the two cleavage sites in prothrombin. A series of approaches will assess the role of the transition between zymogen and proteinase that accompanies the initial cleavage of prothrombin in regulating presentation of the tethered substrate to the enzyme and in providing a rate-limiting step in the process of thrombin formation. Novel fluorescent derivatives of the interacting species will be used in resonance energy transfer studies to establish physical correlates of changes in substrate presentation following proteinase formation and the role of substrate-cofactor interactions in enzymic function. Binding studies will establish the thermodynamic basis for the kinetic behavior of prothrombin as a compound substrate for prothrombinase. These approaches will assess the role of geometric constraints arising from exosite tethering that permit the enzyme to discriminate between the two cleavage sites in the substrate. Additional studies will assess the role of the Nterminal domain of the substrate in participating in the exosite-dependent tethering process. Mechanistic insights obtained under defined conditions will be extended to understand the function of prothrombinase on activated platelets and endothelial cells with the hypothesis that the cleavage pathway and intermediate produced is dependent on cell type, determined by the ability of the cells to support prothrombin binding. Overall, our strategies are based on novel concepts derived from an expanded understanding of substrate recognition by prothrombinase. The proposed approaches will shed new light on important yet poorly understood facets of the biochemistry and biology of enzyme function relevant to normal hemostasis, in disease states and for therapeutic targeting of this reaction in thrombotic and vascular disease.

血液凝固的特异性蛋白水解活化步骤由丝氨酸蛋白酶催化， 它们彼此同源，并且与消化的原型丝氨酸蛋白酶同源。我们将使用凝血酶原 凝血酶原酶复合物催化的活化作为特定大分子底物的范例 通过凝血复合物的识别和切割来研究酶功能的基础。我们的研究是 集中在底物和酶之间的扩展相互作用的核心作用，在驱动的行动， 在凝血酶原的两个裂解位点上的凝血酶原酶。一系列的方法将评估 酶原和蛋白酶之间的转变，伴随着凝血酶原的初始裂解，调节 将所述系链底物呈递给所述酶，并在所述酶的制备过程中提供限速步骤， 凝血酶形成。新的荧光衍生物的相互作用物种将用于共振能量 转移研究，以确定蛋白酶作用后底物呈递变化的物理相关性 形成和酶功能中底物-辅因子相互作用的作用。结合研究将确定 凝血酶原作为凝血酶原酶的复合底物的动力学行为的热力学基础。这些 方法将评估由外部位点束缚引起的几何约束的作用，这些约束允许酶 区分底物中的两个切割位点。其他研究将评估N末端的作用， 域的基板参与外来依赖性拴系过程。机械的见解 在规定的条件下获得的将被扩展到了解凝血酶原酶的功能， 血小板和内皮细胞，假设裂解途径和产生的中间体是 这取决于细胞类型，由细胞支持凝血酶原结合的能力决定。总的来说，我们的 战略是基于新的概念，从扩大理解基板识别， 凝血酶原酶所提出的方法将为人类社会重要但知之甚少的方面提供新的视角。 与疾病状态下的正常止血和治疗相关的酶功能的生物化学和生物学 在血栓性和血管性疾病中靶向该反应。