Physiological Role of Fibrin Anti-Thrombin I Activities

描述(申请人提供)：凝血酶与纤维蛋白原在其 中心E结构域的底物识别位点导致蛋白质降解 纤维蛋白原转化为纤维蛋白。纤维蛋白本身具有明显的 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，其中一类是E 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 人纤维蛋白原中的高亲和力凝血酶结合部位 仅存在于一种称为伽马‘的伽马链变体中，它包含一种独特的 从残基408到427的C末端序列(y‘L-427L)，也与血浆结合 因子XIII。Y‘1-427L上的凝血酶结合位点包含残基414至 427,418和422的酪氨酸硫化，以及最终的三肽 序列(DDL)，对于凝血酶结合是重要的。我们认为最重要的是 血液中抗凝血酶I活性的重要生理方面是 凝血酶与纤维蛋白的无底物结合形成内凝血酶 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的凝血酶作用。 这项建议的目标是通过调查 小鼠纤维蛋白中非底物凝血酶结合位点的作用 人类抗凝血酶I活性的代表。为了实现这一目标，我们将 制备转基因小鼠，其中(1)非凝血酶结合小鼠 Y‘链序列已被凝血酶结合的人Y’序列所取代 (HU-Y‘纤维蛋白原)，或(2)低亲和力凝血酶结合部位 在BB68位突变，产生纤维蛋白原和两种缺陷的凝血酶 底物识别和低亲和力非底物凝血酶结合[那不勒斯I (BB A68T)]。在目标1中，我们将表征Hu-y‘的生化性质。 纤维蛋白(原)，确定引入Hu-y‘on的体内后果 血栓形成、凝血因子XIII介导的交联和纤溶，并进行 血栓调节蛋白缺陷小鼠(TM PRO、TM FLOX)与TO的相互作用 评估Hu-y表型是否改善或加重血栓前病变 或者明显的血栓状态。我们还将对内容物和体内进行评估 完整Y‘1-427L链向非凝血酶结合的代谢转化 (DES-EDDL)y‘1-423P链，使用已开发的ELISA法 用来测量人类的Y链。在目标2中，我们将描述生物化学 小鼠那不勒斯I纤维蛋白(原)的性质，体内后果的评估 在血栓形成方面引入那不勒斯I号突变，并进行 血栓前TM亲小鼠相互作用筛选确定那不勒斯 I表型加剧了高凝状态。我们希望解决任何 关于人类y‘链是有益还是有益的争论 体内对血栓形成或纤溶的有害影响。通过比较 对胡-y‘和那不勒斯I表型的分析，我们将能够定义 抗凝血酶I高亲和力成分和低亲和力成分在血管紧张素转换酶中的作用 血栓的预防和引发。

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.