Hirudisins. Hirudin-derived thrombin inhibitors with disintegrin activity

• Published in: The Journal of biological chemistry 1992-12, Vol.267 (34), p.24230-24234
• Main Authors: KNAPP, A, DEGENHARDT, T, DODT, J
• Format: Article
• Language: English
• Subjects: Adenosine Diphosphate - pharmacology; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Disintegrins; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hirudins - analogs & derivatives; Hirudins - genetics; Hirudins - pharmacology; Humans; Hydrolases; Kinetics; Mathematics; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligopeptides - pharmacology; Peptide Fragments - pharmacology; Peptides - pharmacology; Platelet Aggregation - drug effects; Platelet Aggregation Inhibitors - pharmacology; Platelet Membrane Glycoproteins - isolation & purification; Platelet Membrane Glycoproteins - metabolism; Protein Engineering; Prothrombin - isolation & purification; Recombinant Proteins - pharmacology; Thrombin - antagonists & inhibitors; Venoms - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)35754-5

Recombinant hirudin variants have been designed which inhibit alpha-thrombin by the hirudin mechanism and which in addition exhibit disintegrin activity. These proteins, called "hirudisins," have been engineered by replacing the Ser-Asp-Gly-Glu sequence at the tip of hirudin's finger-like structure (residues 32-35) by Arg-Gly-Asp-Ser (RGDS) to yield hirudisin and Lys-Gly-Asp-Ser (KGDS) to obtain hirudisin-1. Comparison of thrombin inhibition activities showed that hirudisin is 2-fold more potent (K(i) = 160 +/- 70 fM) than hirudisin-1 (K(i) = 370 +/- 44 fM) and recombinant (r)-hirudin (K(i) = 270 +/- 50 fM). alpha-Thrombin-stimulated platelet aggregation was effectively inhibited by r-hirudin, hirudisin, and hirudisin-1 with IC50 of 5.7 to 6.8 nM. Unlike r-hirudin, hirudisin inhibits ADP-induced platelet aggregation (IC50 = 65 microM) 3- to 5-fold stronger than the linear GRGDS- and RGDS-peptide. Direct interaction of hirudisin with purified glycoprotein IIb-IIIa demonstrated that antiplatelet aggregation activity is due to the integrin-directed RGD motif. Disintegrin activity of hirudisin relative to that of reduced and carboxymethylated hirudisin suggests that the conformational strain favors binding to integrins. On the basis of these results, hirudisins appear to be interesting molecules for the design of potential antithrombotic agents with antithrombin as well as antiplatelet aggregation activities.