Structural Correlates of Protease and Cofactor Function

蛋白酶和辅因子功能的结构相关性

• 批准号: 7663367
• 负责人: Rodney M Camire
• 金额: $ 37.93万
• 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/7663367
• 关键词: Active Sites; Allosteric Regulation; Animals; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Biological Models; Biology; Blood Circulation; Blood Clot; Blood Vessels; Blood coagulation; Bypass; Catalytic Domain; Cells; Chemistry; Coagulation Process; Complex; Data; Development; Enzymatic Biochemistry; Enzyme Precursors; Enzymes; Event; Factor Va; Factor X; Factor Xa; Family; Fluorescence Resonance Energy Transfer; Funding; Generations; Hemophilia A; Hemorrhage; Hemostatic function; In Vitro; Infusion procedures; Injury; Instruction; Investigation; Knowledge; Life; Measures; Modeling; Molecular; Multienzyme Complexes; Mus; Mutagenesis; Nature; Pathway interactions; Peptide Hydrolases; Peptides; Phenotype; Physiological; Physiological Processes; Plasma; Play; Principal Investigator; Process; Property; Protease Domain; Protease Inhibitor; Protein Precursors; Proteins; Prothrombin; Resistance; Role; Series; Serine Protease; Site; Specificity; Stimulus; Structure; Techniques; Testing; Therapeutic; Thrombin; Thromboplastin; Thrombosis; Variant; Work; base; cofactor; enzyme substrate; in vitro Model; in vivo Model; inhibitor/antagonist; insight; programs; prothrombin activator; prothrombinase complex; translational study

Program Director/Principal Investigator (Last, First, Middle): KrJShnaSWamy, Sriram PROJECT SUMMARY(See instructions): The serine proteinase factor Xa (FXa) and the cofactor factor Va (FVa) are essential components of the enzymatic complex prothrombinase, the only known physiological activator of prothrombin. A major factor by which prothrombinase achieves its remarkable specificity is through exosites interactions between enzyme, substrate and cofactor. Many of these binding sites are not functional on the precursor of these proteins and the mechanism by which they are conformationally activated is not well understood. Additionally, there are still major gaps in the current understanding of how these sites are allosterically modulated and to what extent these interactions contribute to the enhanced function of the enzyme complex. The broad long-term objective of this work is to decipher these molecular processes and provide detailed mechanistic insight into FXa and FVa function within prothrombinase. In the first aim, we will investigate the role of the FX zymogen to protease transition pathway in macromolecular binding site expression within the catalytic domain. We hypothesize that intramolecular activation of FX following conformational activation of the protease plays a major role in the exposure of substrate, cofactor, and inhibitor binding sites. In the second aim, we will exploit unique FVa variants to examine prothrombin binding and examine the role this interaction plays in prothrombinase function. We hypothesize that specific acidic sequences within the heavy chain of FVa are important for thrombin binding but do not play a major role in prothrombin recognition. In the final aim, we will use FXa "zymogen-like" variants which can be thermodynamicallyrescued by FVa to examine whether they provide effective hemostasis using in vitro and in vivo models of hemophilia. We hypothesize that these variants will be resistant to protease inhibitors and be long lived in the circulation but promote thrombin generation in the presence of FVa. We will test whether these derivatives will be useful therapeutic procoagulant bypass agents in hemophilic animals. Understanding the molecular events leading to the expression of structural determinants on FXa and FVa as well as how these sites influence each other may prove useful in developing pharmacological agents directed at regulating thrombin generation. RELEVANCE (See instructions): The proteins and cells that form a blood clot must remain in a quiescent state and only become activated following injury. This proposal seeks to better understand how one of these proteins, factor X is converted to the active form FXa. The mechanism underlying this process has relevance for better understanding and possibly treating the pathological states of hemophilia and thrombosis. PROJECT/

项目负责人/主要研究者（最后，第一，中间）：KrJShnaSWamy，Sriram 项目总结（见说明）： 丝氨酸蛋白酶因子Xa（FXa）和辅因子Va（FVa）是蛋白酶的必需组分。 酶促复合物凝血酶原酶，唯一已知的凝血酶原生理激活剂。一个主要因素是， 该凝血酶原酶通过酶之间的外切位点相互作用实现其显著的特异性， 底物和辅因子。这些结合位点中的许多在这些蛋白质的前体上不起作用， 它们被构象活化的机理还不清楚。另外存在 目前对这些位点如何被变构调节以及如何被变构调节的理解仍然存在重大差距。 这些相互作用有助于增强酶复合物的功能。广泛的长期 这项工作的目的是破译这些分子过程，并提供详细的机制洞察， FXa和FVa在凝血酶原酶内起作用。在第一个目标中，我们将研究FX酶原的作用 以蛋白酶过渡途径在大分子结合位点内表达催化结构域。我们 假设蛋白酶构象活化后FX分子内活化 在底物、辅因子和抑制剂结合位点的暴露中起主要作用。第二个目标，我们将 利用独特的FVa变体来检查凝血酶原结合，并检查这种相互作用在 凝血酶原酶功能我们假设FVa重链内的特异性酸性序列是 对凝血酶结合很重要，但在凝血酶原识别中不起主要作用。在最后的目标，我们 将使用FXa "酶原样"变异体，这种变异体可以被FVa拯救， 它们使用血友病的体外和体内模型提供有效止血。我们假设这些 变体将对蛋白酶抑制剂具有抗性， 在FVa的存在下产生。我们将测试这些衍生物是否有治疗作用 血友病动物中的促凝血旁路剂。了解导致 FXa和FVa的结构决定因子的表达以及这些位点如何相互影响可能 证明可用于开发针对调节凝血酶生成的药理学试剂。 相关性（参见说明）： 形成血凝块的蛋白质和细胞必须保持静止状态，只有在激活时才会被激活。 受伤后。这项提案旨在更好地了解这些蛋白质之一，因子X是如何转化为 活性形式FXa。这一过程的机制有助于更好地理解和 可能治疗血友病和血栓形成的病理状态。 项目/