Physiological Role of Fibrin Anti-Thrombin I Activities

纤维蛋白抗凝血酶 I 活性的生理作用

• 批准号: 6612843
• 负责人: Michael W Mosesson
• 金额: $ 34.21万
• 依托单位: VERSITI WISCONSIN, INC.
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6612843
• 关键词: antithrombins; binding sites; coagulation factor VIII; crosslink; enzyme linked immunosorbent assay; fibrin; fibrinogen; fibrinolysis; gene mutation; genetically modified animals; laboratory mouse; mutant; protein binding; protein sequence; protein structure function; proteolysis; thrombin; thrombomodulin; thrombosis

DESCRIPTION (provided by applicant): Thrombin binding to fibrinogen at its substrate recognition sites in the central E domain leads to proteolytic conversion of fibrinogen to fibrin. Fibrin itself has an appreciable non-substrate thrombin binding potential termed 'anti-thrombin I', that is defined by two classes of thrombin-binding sites, one of low affinity in the E domain, and the other of high affinity in certain D domains. The low affinity sites represent a residual aspect of fibrinogen substrate binding, whereas the high affinity thrombin binding site in human fibrin(ogen) is situated exclusively in a gamma chain variant termed gamma', which contains a unique C-terminal sequence from residue 408 to 427 (y' l-427L) that also binds plasma factor XIII. The thrombin binding site on y' 1-427L encompasses residues 414 to 427, and Tyr sulfation at 418 and 422 as well as the ultimate tripeptide sequence (DDL), are important for thrombin binding. We believe that the most important physiological aspect of anti-thrombin I activity in blood is that non-substrate thrombin binding to fibrin sequesters thrombin within the forming clot, removing it from the thrombin-generating environment, thereby reducing thrombin feedback clotting activation as well as other direct thrombin effects. The goal of this proposal is to evaluate that hypothesis by investigating the role of non-substrate thrombin binding sites in murine fibrin that are representative of human anti-thrombin I activity. To accomplish this we will prepare genetically-altered mice in which (1) the non-thrombin-binding murine y' chain sequence has been replaced by the thrombin-binding human y' sequence (hu-y' fibrinogen), or (2) the low-affinity thrombin-binding site has been mutated at BB 68 to create fibrinogen with both defective thrombin substrate-recognition and low-affinity non-substrate thrombin-binding [Naples I (BB A68T)]. In Aim 1 we will characterize the biochemical properties of hu-y' fibrin(ogen), determine the in vivo consequences of introducing hu-y' on thrombosis, factor XIII-mediated crosslinking, and fibrinolysis, and carry out an interaction screen in thrombomodulin-deficient mice (TM pro, TM flox) to assess whether the hu-y' phenotype ameliorates or exacerbates the prethrombotic or flagrantly thrombotic state. We will also evaluate the content and in vivo metabolic conversion of intact y' 1-427L chains to non-thrombin-binding (des-EDDL) y' 1-423P chains, using ELISA methodology that has been developed for measuring human y' chains. In Aim 2 we will characterize the biochemical properties of murine Naples I fibrin(ogen), evaluate the in vivo consequences of introducing the Naples I mutation on thrombosis, and carry out an interaction screen in prethrombotic TM pro mice to determine whether the Naples I phenotype exacerbates the hypercoagulable state. We expect to resolve any controversy that now exists as to whether human y' chains have beneficial or deleterious in vivo effects on thrombosis or fibrinolysis. By comparing analyses on the hu-y' and Naples I phenotypes, we will be able to define the specific roles of the high- and low-affinity components of anti-thrombin I in the prevention and initiation of thrombosis.

性状（由申请方提供）：凝血酶与纤维蛋白原结合， 在中央E结构域的底物识别位点导致蛋白水解 纤维蛋白原转化为纤维蛋白。Fiorium本身有一个可观的 非底物凝血酶结合潜力，称为"抗凝血酶I"，即 由两类凝血酶结合位点定义，一类在E 结构域，另一个在某些D结构域中具有高亲和力。低亲和力 位点代表纤维蛋白原底物结合的残留方面，而 人纤维蛋白（原）中高亲和力凝血酶结合位点位于 仅在称为γ '的γ链变体中，其含有独特的 也结合血浆的来自残基408至427的C-末端序列（y 'l-427L） 因子XIII。γ 1 - 427 L上的凝血酶结合位点包括残基414至416。 427处的Tyr硫酸化，418和422处的Tyr硫酸化以及最终的三肽 序列（SEQ ID NO：1）对于凝血酶结合是重要的。我们相信， 血液中抗凝血酶I活性重要生理学方面是， 非底物凝血酶与纤维蛋白结合， 凝块，将其从凝血酶生成环境中移除，从而减少 凝血酶反馈凝血激活以及其他直接凝血酶效应。 本提案的目标是通过调查 非底物凝血酶结合位点在鼠纤维蛋白中的作用 代表人抗凝血酶I活性。为了实现这一目标，我们将 制备遗传改变的小鼠，其中（1）非凝血酶结合鼠 y '链序列已被凝血酶结合人y'序列取代 (hu-y纤维蛋白原），或（2）低亲和力凝血酶结合位点已被 在BB 68突变产生纤维蛋白原， 底物识别和低亲和力非底物凝血酶结合[Naples I (BB A68T）]。在目的1中，我们将表征hu-y '的生物化学性质。 纤维蛋白（原），确定引入Hu-Y 'on的体内结果 血栓形成、因子XIII介导的交联和纤维蛋白溶解， 在血栓调节蛋白缺陷小鼠（TM pro，TM flox）中进行相互作用筛选， 评估hu-y '表型是否改善或加重血栓前病变 或明显的血栓状态。我们还将评估内容和体内 完整γ ′ 1 - 427L链向非凝血酶结合的代谢转化 （des-EDDL）γ 1 - 423 P链，使用已经开发的ELISA方法， 用来测量人类的Y链在目标2中，我们将描述 小鼠Naples I纤维蛋白（原）的性质，评价体内结果 引入那不勒斯I突变对血栓形成，并进行一项 在血栓前TM pro小鼠中进行相互作用筛选，以确定Naples I表型加重高凝状态。我们希望解决任何 目前存在的关于人类Y '链是否具有有益或 对血栓形成或纤维蛋白溶解的有害体内作用。通过比较 通过对hu-y '和Naples I表型的分析，我们将能够定义 抗凝血酶I的高亲和力和低亲和力组分在 预防和引发血栓形成。