Kinetics of human thrombin inhibition by two novel peptide inhibitors (Hirunorm IV and Hirunorm V)

• Published in: Biochemical pharmacology 1996-10, Vol.52 (8), p.1141-1146
• Main Authors: Cappiello, Mario, Vilardo, Pier Giuseppe, Lippi, Annalisa, Criscuoli, Marco, Corso, Antonella Del, Mura, Umberto
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Biological and medical sciences; Blood. Blood coagulation. Reticuloendothelial system; Chromogenic Compounds; coagulation; hirudin; Hirudins - analogs & derivatives; Hirudins - pharmacology; Hirulog; Hirunorm; Humans; In Vitro Techniques; Kinetics; Medical sciences; Molecular Sequence Data; Oligopeptides; Peptide Fragments - pharmacology; Peptides - chemistry; Peptides - pharmacology; Pharmacology. Drug treatments; Proteins - chemistry; Proteins - pharmacology; Recombinant Proteins - pharmacology; Serine Proteinase Inhibitors - chemistry; Serine Proteinase Inhibitors - pharmacology; Substrate Specificity; thrombin; Thrombin - antagonists & inhibitors; thrombin inhibition
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/0006-2952(96)00388-7

A study on the kinetics of human thrombin inhibition by two novel synthetic peptides (Hirunorm IV and Hirunorm V) and a comparison with recombinant hirudin and a commonly used thrombin inhibitor, Hirulog-1, are reported. The dissociation constants for Hirunorm IV and Hirunorm V were determined by varying the concentration of inhibitors at fixed concentrations 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 dependence on the concentration of substrate; this finding, which indicates that the inhibition was competitive, made possible the estimation of the dissociation constants ( K I ) for Hirunorm IV and Hirunorm V, which were 0.134 ± 0.014 nM and 0.245 ± 0.016 nM, respectively. Similar dissociation constants were also obtained for the two inhibitors when thrombin activity was measured with fibrinogen in the clotting assay. When tested for resistance to thrombin proteolytic activity, both inhibitors were inviolate to cleavage by thrombin. The data obtained demonstrate that both Hirunorm IV and Hirunorm V are potent and stable inhibitors of human thrombin activity.