Fibrin(ogen) Structure and Interactions

描述（由申请人提供）：纤维蛋白原是一种多结构域多功能血浆蛋白，在凝血酶介导转化为纤维蛋白后，在许多重要的生理和病理过程中起着突出作用，包括止血、血栓形成、伤口愈合、炎症、血管生成和动脉粥样硬化。为了更好地理解这些过程的机制，需要建立这种复杂分子的结构和相互作用。我们研究的长期目标是全面了解纤维蛋白原和纤维蛋白的结构，并建立它们与各种蛋白质和细胞类型多重相互作用的分子机制。近十年来，对纤维蛋白原结构的研究取得了重大进展。然而，其A1链的c端部分（1C-结构域）和B2链的n端部分（b2n -结构域）的结构组织尚不清楚。大量数据表明，纤维蛋白原中1C结构域的构象可能与纤维蛋白中的构象不同，在纤维蛋白中它们形成1C-聚合物并变得高活性。尽管如此，c -聚合物形成的分子机制仍有待确定。该应用主要关注1c结构域，它参与纤维蛋白组装、纤维蛋白依赖性纤维蛋白溶解、血管生成、动脉粥样硬化和肾淀粉样变性。第一个Specific Aim是建立分离纤维蛋白原1c结构域的NMR溶液结构，阐明该结构域在纤维蛋白1c聚合物中的结构组织，阐明由1c结构域区域突变引起的遗传性肾淀粉样变性的结构基础。第二个特定目标是阐明1c结构域分子内和分子间相互作用的分子机制，这些结构域负责纤维蛋白中生理活性1c聚合物的形成，以及它们获得性和先天性缺陷的病理后果。这些目标将通过制备重组1c结构域片段及其剪切变体，以及重组和蛋白学获得的代表纤维蛋白（原）中心区域和(B) 2n结构域的片段，并通过生化和生物物理方法研究它们的结构和众多相互作用来实现。拟议的研究将产生基础知识，这将对我们理解正常和病理条件下纤维蛋白凝块形成的分子机制以及控制上述纤维蛋白依赖过程的能力产生影响。由于c结构域的获得性和先天性缺陷可能导致严重的病理后果，包括出血、各种血栓性疾病和肾淀粉样变性，因此拟议的研究也将进一步阐明这些病理的分子机制，从而有助于其治疗的发展。公共卫生相关性：拟议的研究将产生关于纤维蛋白（原）单个结构域的结构和功能的基本知识，纤维蛋白（原）是凝血级联的主要成分。这些知识需要更好地理解和控制纤维蛋白（原）依赖过程的分子机制，如血液凝固、纤维蛋白溶解、炎症和血管生成。由于这些结构域的先天性和获得性缺陷可能导致严重的病理后果，包括出血、各种血栓性疾病和肾淀粉样变性，因此拟议的研究将进一步阐明这些病理的分子机制，从而有助于其治疗的发展。

DESCRIPTION (provided by applicant): Fibrinogen is a multidomain polyfunctional plasma protein that after thrombin-mediated conversion into fibrin plays a prominent role in a number of important physiological and pathological processes, including hemostasis, thrombosis, wound healing, inflammation, angiogenesis, and atherogenesis. Better understanding of the mechanisms underlying these processes requires establishment of the structure and interactions of this complex molecule. The long-term objectives of our research are to generate comprehensive knowledge on the structure of fibrinogen and fibrin and to establish the molecular mechanisms of their multiple interactions with various proteins and cell types. Significant progress has been made in understanding the fibrinogen structure in the last decade. However, the structural organization of the C-terminal portions of its A1 chains (1C- domains) and the N-terminal portions of its B2 chains (B2N-domains) is still unclear. Numerous data suggest that the conformation of the 1C-domains in fibrinogen may differ from that in fibrin, in which they form 1C- polymers and become highly reactive. Still, the molecular mechanisms of 1C-polymer formation remain to be established. This application focuses on the 1C-domains, which are involved in fibrin assembly, fibrin- dependent fibrinolysis, angiogenesis, atherogenesis, and renal amyloidosis. The first Specific Aim is to establish the NMR solution structure of the isolated fibrinogen 1C-domain, to elucidate the structural organization of this domain in fibrin 1C-polymers, and to clarify the structural basis for hereditary renal amyloidosis caused by mutations in the 1C-domain region. The second Specific Aim is to elucidate the molecular mechanisms of the intra- and intermolecular interactions of the 1C-domains, which are responsible for the formation of physiologically active 1C-polymers in fibrin, and for the pathological consequences of their acquired and congenital defects. These aims will be accomplished by preparation of recombinant 1C-domain fragments and their truncated variants, as well as recombinant and proteolitically obtained fragments that represent the central region of fibrin(ogen) with the (B)2N-domains, and studying their structure and numerous interactions by biochemical and biophysical methods. The proposed studies will generate basic knowledge that will have an impact on our understanding of the molecular mechanisms underlying formation of fibrin clots in normal and pathological conditions and ability to control the above mentioned fibrin-dependent processes. Because acquired and congenital defects in the 1C-domains may cause severe pathological consequences, including hemorrhage, various thrombotic disorders, and renal amyloidosis, the proposed studies will also further clarify the molecular mechanisms underlying these pathologies, and thereby contribute to the development of their therapies. PUBLIC HEALTH RELEVANCE: The proposed studies will generate basic knowledge about the structure and function of individual domains of fibrin(ogen), the major component of the blood coagulation cascade. Such knowledge is required to better understand and control the molecular mechanisms underlying the fibrin(ogen)-dependent processes, such as blood clotting, fibrinolysis, inflammation, and angiogenesis. Because congenital and acquired defects in these domains may cause severe pathological consequences, including hemorrhage, various thrombotic disorders, and renal amyloidosis, the proposed studies will further clarify the molecular mechanisms underlying these pathologies, and thereby contribute to the development of their therapies.