Thrombin-specific inhibition by and slow cleavage of hirulog-1

Abstract

Hirulog-1 {D-Phe-Pro-Arg-Pro-[Gly]4-desulphohirudin-(53-64) (HV1)} was designed to bind by its first four and last 12 residues to the α-thrombin catalytic site and anion-binding exosite for fibrin(ogen) recognition respectively, with a [Gly]4 bridge and an Arg-Pro bond at the scissional position. Human α-, γ- and ζ-thrombins, as well as bovine trypsin, readily hydrolyse Spectrozyme-TH (D-hexahydrotyrosyl-Ala-Arg p-nitroanilide) at pH 7.4 and approx. 23°C. Both α- and ζ-thrombins, which have high fibrinogen-clotting activities (> 3000 kunits/g), were inhibited with this substrate by hirulog-1 [K(i) = 2.56 ± 0.35 nM (n = 3) and 1.84 ± 0.15 nM (n = 3) respectively] and slowly cleaved the inhibitor [k = 0.326 ± 0.082 min-1 (n = 12) and 0.362 ± 0.056 min-1 (n = 18) respectively], whereas γ-thrombin which has essentially no clotting activity (approx. 4 kunits/g), and trypsin were not inhibited with > 1000-fold molar excess of hirulog-1. Similar inhibition parameters were also obtained for hirulog-1 incubated with α-thrombin or ζ-thrombin at approx. 23°C and by measuring thrombin activity with fibrinogen in the clotting assay at 37°C. Cleavage of the Arg-3-Pro-4 bond in hirulog-1 by either α- or ζ-thrombin was shown by identical cleavage products of either thrombin on h.p.l.c. and by sequence analysis of the α-thrombin products. These data demonstrate that hirulog-1 is a specific inhibitor of thrombin forms with high fibrinogen-procoagulant activities and that its Arg-3-Pro-4 bond is slowly cleaved by these thrombin forms.