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.

该项目的重点是一种假设，即血栓的形成是 由互补的吸引力所产生的力量驱动 位于氨基或羧基末端结构域的聚合中心 纤维蛋白分子。有人建议澄清哪种氨基酸 纤维蛋白的三条多肽链中的每一条都需要序列来 表达与纤维蛋白凝块的结合亲和力。含有序列的多肽 Alpha链和Beta链氨基末端，以及Bty链和Gamma链 通过对纤维蛋白原的酶促裂解，可以得到羧基末端 和纤维蛋白与各种蛋白酶或通过化学合成。初步 数据显示与纤维蛋白的结合亲和力依赖于完整的构象 片段d1和t-NDSK。据推测，A的结构 聚合点可以包含一个以上的多肽链片段。 为了证明这一点，纤维蛋白原和纤维蛋白片段将被交联。 使用双功能试剂，产物被蛋白酶切割，以及 具有与纤维蛋白亲和力的多链衍生物分离和 特色化的。将测量是否交联型多链 衍生物对血栓的亲和力比简单的多肽更高。 通过纤维蛋白结合区分的多肽将被用来获得单克隆 针对聚合位点的抗体。这些抗体将用于 分离特定聚合位点并绘制上的位点图 纤维蛋白碎片。纤维蛋白聚合点将用于 构建纤溶杂交体。这种混合体将含有一种分子 证明对纤维蛋白有亲和力的载体，例如片段DD或E1， 以非共价或共价方式与纤溶剂连接， 尤其是使用组织型纤溶酶原激活剂和尿激酶剂。初步 结果表明，含有片段DD和(DD)E的杂交体中的激活剂为 保护血液免受血液抑制。这种效果通过放大来表达。 血浆凝块的纤溶作用。实验结果将提供一种 纤溶杂交物作为血栓靶向的有效性的证据 物种。血浆和全血凝块的使用将允许量化 各种杂交种的溶解效果。该计划的长远目标 该提案将对参与的分子机制提供解释 形成血栓，开发新型抗凝剂 聚合位结构与溶栓新途径的探索 心理治疗。