Clot-bound thrombin is functionally active and relatively protected from heparin-ATIII inhibition. It may contribute to re-thrombosis after thrombolysis and the propagation of deep venous thrombosis. A library of thrombin mutants generated by alanine scanning mutagenesis was used to map thrombin's fibrin binding site. Thrombin mutants were used to compete with 125I-wild type (WT) thrombin binding to fibrin clot. Six mutants involving eight residues were relatively defective in competition, suggesting that these residues are important for interaction with fibrin. Mapping these residues onto thrombin's surface shows that they are clustered together on a surface distinct from the exosites I and II, supporting the hypothesis that the fibrin-binding site is mediated by a discrete domain.
Specific Aim number 1. Mapping the fibrin-binding site on thrombin Selected mutants with significantly diminished capability to displace WT thrombin will be expressed and direct binding to fibrin clot characterized. Additional alanine mutants involving neighboring residues will be made to more precisely define the fibrin-binding site.
Specific Aim number 2. Development of peptides that will displace thrombin from fibrin clot By identifying the specific fibrin-binding site, we will synthesize peptides that will displace thrombin from fibrin clot. Both linear and cyclized peptides will be tested and the tertiary structure of the most potent peptide determined by NMR spectroscopy. Their efficacy as a new class of antithrombotic which has no intrinsic direct or indirect thrombin inhibition but will utilize plasma ATIII in the presence or absence of heparin to inhibit the displaced thrombin will be tested in a rabbit thrombosis model in vivo.
Specific Aim number 3. Testing the fibrin-binding deficient thrombin in a mouse model Recently Dr. Sandra Degen has successfully generated hemizygous and homozygous prothrombin deficient mice using ES cell/gene targeting technology. In collaboration with Dr. Degen, we will create transgenic mice expressing the fibrin-binding deficient prothrombin mutant (II+/+, TG+/-) and crossing with hemizygous prothrombin deficient (II+/-) mice to obtain mice null for the endogenous prothrombin gene but carrying the mutant transgene (II-/-, TG+/-). These mice will be tested in systemic as well as local venous thrombosis models. These studies will provide important structural and functional information on thrombin's fibrin binding site and help to define the role of clot bound thrombin in vivo. It may also lead to the development of novel antithrombotic agents and new thrombosis models in the mouse.
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