KINETICS OF HUMAN THROMBIN INHIBITION BY 2 NOVEL PEPTIDE INHIBITORS (HIRUNORM-IV AND HIRUNORM-V)

A study on the kinetics of human thrombin inhibition by two novel synt hetic peptides (Hirunorm IV and Hirunorm V) and a comparison with reco mbinant hirudin and a commonly used thrombin inhibitor, Hirulog-1, are reported. The dissociation constants for Hirunorm TV and Hirunorm V w ere determined by varying the concentration of inhibitors at fixed con centrations of the chromogenic substrate Chromozym-TH (N-tosylglycyl-L -prolyl-L-arginine 4-nitroanilide acetate). Both inhibitors behaved as reversible tight-binding inhibitors of amidolytic thrombin activity. The apparent dissociation constants determined showed a linear depende nce on the concentration of substrate; this finding, which indicates t hat the inhibition was competitive, made possible the estimation of th e dissociation constants (K-I) for Hirunorm IV and Hirunorm V, which w ere 0.134 +/- 0.014 nM and 0.245 +/- 0.016 nM, respectively. Similar d issociation constants were also obtained for the two inhibitors when t hrombin activity was measured with fibrinogen in the clotting assay. W hen tested for resistance to thrombin proteolytic activity, both inhib itors were inviolate to cleavage by thrombin. The data obtained demons trate that both Hirunorm IV and Hirunorm V are potent and stable inhib itors of human thrombin activity.