THROMBIN INTERACTIONS WITH FIBRIN AND FIBRINOGEN

我们的长期目标是了解每一个生物功能， 纤维蛋白原和纤维蛋白。我们提出这项建议的具体目的是， 并表征凝血酶-纤维蛋白和凝血酶-纤维蛋白原结合 交互.凝血酶与纤维蛋白的结合是通过其外部位点发生的， “非底物”位点，以及纤维蛋白原中的“底物识别”位点 绝佳的价钱我们将研究非底物凝血酶结合位点， 纤维蛋白，其中之一是低亲和力，位于附近或毗邻 在E结构域中具有纤维蛋白原底物识别位点（Aim 1）。 另一个高亲和力的位点位于羧基末端， γ '链区域，是正常的次要（约10%）γ 链变体。使用纤维蛋白凝块系统来测量凝血酶结合，我们 将量化在每种类型的凝血酶结合位点的相互作用， 纤维蛋白，表征其结构，并确定确切的γ ' 解释凝血酶结合的序列。我们还将确定 凝血酶-γ-肽复合物的晶体结构， 表征凝血酶-γ '链相互作用。几个功能 将研究凝血酶-纤维蛋白结合相互作用的各个方面， 包括：1）凝血酶与纤维蛋白（原）结合对凝血酶的影响 受体介导的血小板聚集和凝血酶受体裂解 在表达凝血酶受体的细胞系统中; 2） 非酶促凝血酶介导的纤维蛋白聚合加速; 3） γ ′序列对纤维蛋白原向纤维蛋白转化的影响， 纤维蛋白聚合物结构; 4）纤维蛋白聚合物的形成机制 抑制含γ '纤维蛋白的纤维蛋白溶解。我们亦会研究 底物识别位点的凝血酶-纤维蛋白原结合相互作用 (Aim 2）使用活性位点抑制的凝血酶（例如，D419 N凝血酶）， 表征和定量凝血酶-纤维蛋白原结合相互作用。 使用天然纤维蛋白原或蛋白水解修饰的纤维蛋白原缺乏 底物识别位点的部分，以及 与纤维蛋白（原）中心E结构域的结构同源，我们 将映射底物识别位点，并与 纤维蛋白（原）的中心E结构域，我们将绘制底物 识别位点，并将其组成结构与那些 包括纤维蛋白中的低亲和力非底物位点。总之，我们的 调查将阐明结构和功能的作用， γ '序列和纤维蛋白中的非底物凝血酶结合位点 E结构域，描绘了纤维蛋白原的组成结构 底物识别位点，并表征和量化 与凝血酶结合。这些研究将有助于我们了解 凝血酶-纤维蛋白（原）相互作用在调节纤维蛋白 凝块形成、临床血栓形成、纤维蛋白溶解和伤口愈合。

Our long range goal is to understand each of the biological functions of fibrinogen and fibrin. Our specific aims in this proposal are to define and characterize thrombin-fibrin and thrombin-fibrinogen binding interactions. Thrombin binding to fibrin occurs through its exosite at 'non-substrate' sites, and also in fibrinogen at a 'substrate recognition' site. We will investigate the non-substrate thrombin-binding sites on fibrin, one of which is of low affinity, located near to or contiguous with the fibrinogen substrate recognition site in the E domain (Aim 1). The other site, of high affinity, is located in the carboxyl-terminal region of gamma' chains, which are normal minor (approximately 10%) gamma chain variants. Using a fibrin clot system to measure thrombin binding, we will quantify the interactions at each type of thrombin binding site in fibrin, characterize their structures, and determine the exact gamma' sequences accounting for thrombin binding. We will also determine the crystal structure of thrombin-gamma' peptide complexes to further characterize the thrombin-gamma' chain interaction. Several functional aspects of the thrombin-fibrin binding interaction will be investigated, including: 1) the effect of thrombin binding to fibrin(ogen) on thrombin receptor-mediated platelet aggregation, and on thrombin receptor cleavage in a cell system expressing the thrombin receptor; 2) the mechanism of non-enzymatic thrombin-mediated acceleration of fibrin polymerization; 3) the effect of the gamma ' sequence on fibrinogen to fibrin conversion, fibrin assembly, and fibrin polymer structure; 4) the mechanism of suppressed fibrinolysis in gamma '-containing fibrin. We will also examine thrombin-fibrinogen binding interactions at the substrate recognition site (Aim 2) using active-site inhibited thrombins (e.g., D419N thrombin) to characterize and quantify the thrombin-fibrinogen binding interaction. Using native fibrinogen or proteolytically modified fibrinogen lacking portions of the substrate recognition site, and peptides that are homologous with structures in the central E domain of fibrin(ogen), we will map the substrate recognition site, and homologous with structures in the central E domain of fibrin(ogen), we will map the substrate recognition site, and compare its constituent structures with those comprising the low affinity non-substrate site in fibrin. In summary, our investigations will elucidate the structural and functional roles of the gamma' sequence and the non-substrate thrombin binding site in the fibrin E domain, delineate the constituent structures comprising the fibrinogen substrate recognition site, and characterize and quantify the mechanics of binding with thrombin. These studies will contribute to our understanding of the roles) of thrombin-fibrin(ogen) interactions in mediating fibrin clot formation, clinical thrombosis, fibrinolysis, and wound healing.