Improvement of the specificity of dipetarudin by site directed mutagenesis

Abstract

Protease specificity is crucial to the design of thrombin inhibitors as inhibition of other physiologically relevant serine-proteases can compromise their clinical use. Dipetarudin, a potent thrombin inhibitor, also inhibits trypsin and plasmin. Due to the specificity of an inhibitor being influenced by the amino acid residue at the P1 position, we replaced the Arg10 at P1 position of dipetarudin by a histidine, which is the P1 residue of rhodniin, a very specific thrombin inhibitor. The amino acid replacement was carried out by site directed mutagenesis. The mutant, dipetarudinR10H, showed a loss of plasmin and trypsin inhibitory activities present in its wild-type counterpart and a 3-fold higher dissociation constant for thrombin than dipetarudin. However, compared to dipetarudin and r-hirudin, dipetarudinR10H showed similar activity in coagulation screening assays such as activated partial thromboplastin time (aPTT), prothrombin time (PT), ecarin clotting time (ECT) and ecarin chromogenic assay (ECA).