FIBRIN POLYMERIZATION SITES--STRUCTURE AND CLOT BINDING

纤维蛋白聚合位点——结构和凝块结合

• 批准号: 3350999
• 负责人: ANDREI Z BUDZYNSKI
• 金额: $ 26.91万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-05-01 至 1991-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3350999
• 关键词: antifibrinolytic agents; chemical binding; chemical chain length; chemical structure function; crosslink; fibrin; fibrinogen; human tissue; monoclonal antibody; plasminogen activator; polymerization; protein engineering; protein sequence; thrombosis; urokinase

The project is focused on a hypothesis that the formation of a clot is driven by forces originating from attraction of complementary polymerization sites located in either amino- or carboxy-terminal domains of the fibrin molecule. It is proposed to elucidate which amino acid sequence, in each of the three polypeptide chains of fibrin, is required to express binding affinity for fibrin clots. Peptides containing sequences of the Alpha and Beta chain amino-termini, and the Bety and Gamma chain carboxy-termini will be obtained either by enzymatic cleavage of fibrinogen and fibrin with various proteinases or by chemical synthesis. Preliminary data show dependence of binding affinity to fibrin on intact conformation of fragment D1 and t-NDSK. It is postulated that the structure of a polymerization site may contain more than one polypeptide chain segment. To prove this point, fibrinogen and fibrin fragments will be crosslinked with bifunctional reagents, the products cleaved by proteinases, and multi-chain derivatives with affinity for fibrin isolated and characterized. It will be measured whether crosslinked multi-chain derivatives possess higher affinity for clots than simple peptides. Peptides distinguished by fibrin binding will be used to obtain monoclonal antibodies against polymerization sites. These antibodies will be used for isolation of specific polymerization sites and for mapping of the sites on fibrin fragments. Fibrin polymerization sites will be utilized to construct fibrinolytic hybrids. The hybrids will contain a molecular vehicle with proven affinity for fibrin, for example fragment DD or E1, linked either non-covalently or covalently with a firbinolytic agent, especially with tissue plasminogen activator and urokinase. Preliminary results show that the activator in hybrids with fragments DD and (DD)E is protected against blood inhibitors. The effect is expressed by amplified fibrinolysis of plasma clots. Experimental results will provide an evidence as to the usefulness of fibrinolytic hybrids as clot-targeted species. The use of plasma and whole blood clots will allow quantification of the lytic effect of various hybrids. The long-term objectives of the proposal will provide explanation of molecular mechanisms involved in the formation of blood clots, develop new anticoagulants based on polymerization site structure and explore novel approach to thrombolytic therapy.

该项目的重点是一个假设，即凝块的形成是 由源自互补的吸引力的力驱动 位于氨基或羧基末端域中的聚合位点 纤维蛋白分子的。 提议阐明哪种氨基酸 在纤维蛋白的三个多肽链中的每一个中，序列需要 对纤维蛋白凝块的表达结合亲和力。 含有序列的肽 Alpha和Beta链氨基末端，以及Bety和Gamma链 羧基末端将通过纤维蛋白原的酶促切割获得 和纤维蛋白具有各种蛋白酶或化学合成。 初步的 数据显示结合亲和力与纤维蛋白对完整构象的依赖性 碎片D1和T-NDSK的。 据推测， 聚合位点可能包含一个以上的多肽链段。 为了证明这一点，将将纤维蛋白原和纤维蛋白片段交联 使用双功能试剂，产物被蛋白酶裂解，并且 对纤维蛋白分离的多链衍生物，并且 特征。 它将测量是否交联的多链 与简单肽相比，衍生物对凝块具有更高的亲和力。 通过纤维蛋白结合区分的肽将用于获得单克隆 抗聚合位点的抗体。 这些抗体将用于 隔离特定的聚合位点和用于映射位点上的位点 纤维蛋白片段。 纤维蛋白聚合位点将用于 构建纤维蛋白水解杂种。 杂种将包含一个分子 具有对纤维蛋白的亲和力的车辆，例如碎片DD或E1， 与Firbinoltic剂无外交或共价链接， 尤其是组织纤溶酶原激活剂和尿激酶。 初步的 结果表明，具有片段DD和（DD）E的杂种中的激活剂为 防止血液抑制剂。 效果通过放大表达 血浆凝块的纤维蛋白解。 实验结果将提供 关于凝块靶向纤维蛋白水解杂种的有用性的证据 物种。 血浆和全血凝块的使用将允许定量 各种杂种的裂解作用。 长期目标 提案将提供有关涉及的分子机制的解释 血块的形成，基于 聚合位点结构并探索溶栓的新方法 治疗。