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.
Showing the most recent 10 out of 18 publications
