Thrombin inhibition by covalently bound hirudin

Abstract

Hirudin is the most potent known natural inhibitor of thrombin and is presently gaining popularity as an anticoagulant since recombinant and synthesized forms have become available. We have made use of recombinant hirudin (rHir) by covalently binding it to both biomolecules and prosthetic biomaterials. Heterobifunctional crosslinking reagents were used to derivatize rHir and form covalent crosslinks between rHir and albumin producing active conjugates. Both derivatized rHir and conjugates inhibited human alpha-thrombin similarly, however, both showed a ten-fold decrease of alpha-thrombin inhibition when compared to rHir alone using the tripeptide substrate, S-2238. Immobilization of 2.75 +/- 0.45 micrograms rHir on 1.0 cm2 Dacron prosthetic graft patches resulted in inhibition of 1.88 +/- 0.03 micrograms alpha-thrombin in solution (mean +/- SD, n = 3), which is a 8:1 molar ratio, respectively. rHir ED50 inhibition of 0.1 NIH U alpha thrombin stimulated whole blood platelet aggregation was 0.12 x 10(-6) microM. The conjugate ED50 inhibition was 1.37 x 10(-6) microM showing an eleven-fold loss of activity. We conclude that there is only a ten-fold loss of inhibitory activity when rHir is covalently immobilized and that this technique has a benefit of localizing antithrombin activity to surfaces or soluble carrier molecules.