THROMBIN INTERACTIONS WITH FIBRIN AND FIBRINOGEN

我们的长期目标是了解人的每一种生物功能 纤维蛋白原和纤维蛋白。我们在这项提案中的具体目标是界定 以及凝血酶-纤维蛋白和凝血酶-纤维蛋白原结合的特征 互动。凝血酶与纤维蛋白的结合是通过其胞外 ‘非底物’位点，也在纤维蛋白原的‘底物识别’ 地点。我们将研究非底物凝血酶结合位点 纤维蛋白，其中一种是低亲和力的，靠近或毗邻 纤维蛋白原底物识别位点位于E结构域(目标1)。 另一个高亲和力的部位位于羧基末端 伽马‘链区域，它是正常的次要伽马(约10%) 链式变种。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部位的相互作用 纤维蛋白，表征它们的结构，并确定确切的伽马 说明凝血酶结合的序列。我们还将确定 凝血酶-γ‘肽复合体的晶体结构研究 表征凝血酶-伽马‘链相互作用。几种功能 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及其对凝血酶受体切割的影响 在表达凝血酶受体的细胞系统中；2)凝血酶受体的作用机制 非酶促凝血酶促进纤维蛋白聚合；3) 伽马序列对纤维蛋白原到纤维蛋白转化的影响， 纤维蛋白组装和纤维蛋白聚合物结构；4)纤维蛋白的作用机制 抑制含有γ‘的纤维蛋白的纤溶作用。我们还将检查 凝血酶-纤维蛋白原结合底物识别位点的相互作用 (目标2)使用活性部位抑制凝血酶(例如D419N凝血酶) 表征和量化凝血酶-纤维蛋白原结合作用。 使用天然纤维蛋白原或缺乏蛋白水解性的纤维蛋白原 底物识别位点的一部分，以及 与纤维蛋白(原)中心E结构域的结构同源，我们 将绘制底物识别位点图，并与 纤维蛋白(原)的中心E结构域，我们将映射底物 识别位点，并将其组成结构与 包括纤维蛋白中的低亲和力非底物部位。总而言之，我们的 研究将阐明细胞的结构和功能作用 γ‘序列与纤维蛋白中的非底物凝血酶结合部位 E结构域，描绘组成纤维蛋白原的组成结构 底物识别部位，并表征和量化的机制 与凝血酶结合。这些研究将有助于我们理解 凝血酶-纤维蛋白(原)相互作用在调节纤维蛋白中的作用 血栓形成、临床血栓形成、纤溶和伤口愈合。

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.