STRUCTURE/FUNCTION OF PROTEIN C

蛋白质 C 的结构/功能

• 批准号: 2029114
• 负责人: JOHN H GRIFFIN
• 金额: $ 44.25万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-12-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2029114
• 关键词: X ray crystallography; activation product; anticoagulants; blood coagulation; calcium; chemical binding; coagulation factor V; complementary DNA; drug resistance; human subject; laboratory mouse; laboratory rabbit; molecular site; monoclonal antibody; mutant; protein C; protein S; protein purification; protein sequence; protein structure function; site directed mutagenesis; synthetic peptide; thrombin; thrombomodulin; thrombosis

This proposal seeks continuation of support for long-term studies of molecules responsible for regulation of thrombosis by the protein C pathway. Hereditary protein C (PC) and protein S deficiencies are associated with thrombotic disease and activated protein C (APC) exhibits potent antithrombotic activity in animal models of thrombosis. APC is a normal circulating component in human blood which probably regulates basal levels of thrombin generation in vivo. Clinical trials of PC concentrates for antithrombotic therapy have been initiated. Plasma from many thrombophilia patients gives a poor anticoagulant response to APC. This property of plasma, called "APC resistance", is the most common identifiable defect among venous thrombophilia patients. This proposal will test the hypothesis, based on our preliminary results, that abnormal factor V is responsible for APC resistance. Another hypothesis to be tested is that molecular mechanisms for the regulation of the anticoagulant activity of PC and APC are determined by specific amino acids on the surface of PC and APC. Thus, the five specific aims include the following. (1) To characterize abnormalities of factor V hypothesized to be responsible for APC resistance, we will sequence patients' factor V cDNA and perform studies involving purification and characterization of normal and patients' factor V to identify abnormalities at the molecular level. (2) To extend analysis of three-dimensional homology models to the entire molecules of PC and APC and to APC:serpin complexes, we will continue development of computer modelling studies to provide models which will be used to explain clinical phenotypes for naturally occurring mutations and to design and interpret structure-function studies. (3) To identify functional roles of specific surface loops of residues of PC and APC, we will use synthetic oligopeptides and polypeptides and antipeptide antibodies to identify essential sites and residues on the surface of PC and APC. Studies will employ synthetic oligopeptides and polypeptides, including among other, synthetic individual N- and C-terminal EGF domains, chemically-ligated polypeptides containing both EGF domains, and synthetic peptides containing the putative Ca2+-binding loop of the protease domain (residues 225-235). (4) To identify functionally essential residues at specific surface sites of PC and APC, we will use site-directed mutagenesis. Studies will focus on amino acids hypothesized to be involved in Ca2+-ion binding by the protease domain and in intermolecular interactions of PC or APC with thrombin:thrombomodulin, factor Va, and protein S. The roles of positive residues in a novel exosite will be probed. New inhibitor-resistant APC mutants will be sought. Studies will include defining the mechanism of anticoagulant action of the inhibitor-resistant mutant, S360A-APC, and using this molecule to study interactions of APC with normal and abnormal factor Va and with protein S. (5) To prepare PC and APC crystals suitable for x-ray diffraction determination of their three-dimensional structures. The proposed studies will provide new knowledge about natural anticoagulant mechanisms and may lead to new approaches to control thrombosis.

该建议寻求继续支持长期研究 负责通过蛋白C调节血栓形成的分子 路径。遗传蛋白C（PC）和蛋白质的缺陷是 与血栓性疾病和活化蛋白C（APC）有关 血栓形成动物模型中有效的抗血栓性活性。 APC是一个 人体血液中可能调节的正常循环成分 体内凝血酶产生的基础水平。 PC的临床试验 已经开始进行抗血栓疗法的浓缩物。来自 许多血小板患者对APC的抗凝反应较差。 血浆的这种属性称为“ APC抗性”，是最常见的 静脉血栓形成患者的可识别缺陷。这个建议 将根据我们的初步结果检验假设，异常 因子V负责APC电阻。另一个假设是 测试的是调节的分子机制 PC和APC的抗凝活性由特定氨基确定 PC和APC表面上的酸。因此，五个具体目标包括 下列。 （1）表征因子V的异常 为了负责APC耐药性，我们将对患者的因子进行测序 V cDNA并进行涉及纯化和表征的研究 正常和患者的因子V的识别异常 分子水平。 （2）扩展三维同源性的分析 模型与PC和APC的整个分子以及APC：SERPIN复合物， 我们将继续开发计算机建模研究以提供 用于解释自然的临床表型的模型 发生突变并设计和解释结构功能 研究。 （3）确定特定表面环的功能作用 PC和APC的残留物，我们将使用合成寡肽和 多肽和抗肽抗体，以识别必需位点和 PC和APC表面的残留物。研究将采用合成 寡肽和多肽，包括合成 单个N-和C末端EGF结构域，化学结合的多肽 包含两个EGF结构域和包含的合成肽 推定的Ca2+结合蛋白酶结构域（残基225-235）。 （4） 在特定的表面位点识别功能上必不可少的残基 PC和APC，我们将使用定位的诱变。研究将重点 假设氨基酸与Ca2+-ion结合参与 蛋白酶结构域以及PC或APC与分子间相互作用与 凝血酶：血小板调节蛋白，因子VA和蛋白质。阳性的作用 将探测一种新型外部的残留物。新型抑制剂APC 将寻求突变体。研究将包括定义机制 抗凝剂耐药物突变体S360A-APC和抗凝作用 使用该分子研究APC与正常和异常的相互作用 因子VA和蛋白质S。（5）准备PC和APC晶体 用于X射线衍射测定其三维 结构。拟议的研究将提供有关自然的新知识 抗凝机制，可能导致新的控制方法 血栓形成。