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FIBRIN POLYMERIZATION SITES--STRUCTURE AND CLOT BINDING

纤维蛋白聚合位点——结构和凝块结合

基本信息

批准号：
3351000
负责人：
ANDREI Z BUDZYNSKI
金额：
$ 26.13万
依托单位：
TEMPLE UNIV OF THE COMMONWEALTH
依托单位国家：
美国
项目类别：
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/3351000
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

项目摘要

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，即血块的形成是由互补的吸引力产生的力驱动的位于氨基或羧基末端结构域的聚合位点纤维蛋白分子。建议阐明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，表达对纤维蛋白凝块的结合亲和力。含肽序列 α链和β链的氨基末端，以及β链和γ链的氨基末端，羧基末端将通过纤维蛋白原的酶裂解获得和纤维蛋白与各种蛋白酶或通过化学合成。初步数据显示对纤维蛋白的结合亲和力依赖于完整构象片段D1和t-NDSK。据推测，一个聚合位点可以含有多于一个多肽链片段。为了证明这一点，纤维蛋白原和纤维蛋白碎片将交联用双功能试剂，产物被蛋白酶裂解，和分离的对纤维蛋白具有亲和性的多链衍生物，表征了将测量交联的多链衍生物比简单肽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体。针对聚合位点的抗体。这些抗体将用于分离特定的聚合位点，并对聚合物上的位点进行定位。纤维蛋白碎片将利用固定聚合位点，构建纤溶杂合体。混合体会包含一种分子对纤维蛋白具有已证实的亲和力的载体，例如片段DD或E1，与纤维蛋白溶解剂非共价或共价连接，尤其是组织纤溶酶原激活剂和尿激酶。初步结果表明，具有片段DD和（DD）E的杂合体中的激活剂是防止血液抑制剂。该效应通过放大的血浆凝块的纤维蛋白溶解。实验结果将提供一个纤溶混合物作为血栓靶向治疗的有用性的证据物种使用血浆和全血凝块将允许定量各种杂交体的裂解作用。的长期目标建议将提供解释的分子机制参与血栓的形成，开发新的抗凝剂的基础上，聚合位点结构和探索新的溶栓途径疗法