Unsulfated N α-acetyl-hirudin45–65 (MDL 27 589), which corresponds to the C-terminus of hirudin1–65, was synthesized by solid-phase methods. The synthetic peptide was able to inhibit fibrin formation and the release of fibrinopeptide A from fibrinogen by thrombin. The catalytic site of thrombin was not perturbed by the synthetic peptide as H-D-Phe-Pip-Arg-pNA hydrolysis (amidase activity) was not affected. The binding of synthetic peptide and thrombin was assessed by isolation of the complex on gel-filtration chromatography. A single binding site with a binding affinity (K a) of approx. 1.0 × 105 M−1 was observed for thrombin-hirudin45–65 interaction. The data suggest that the C-terminal residues 45–65 of hirudin contain a binding domain which recognizes thrombin and yet does not bind to the catalytic site of the enzyme.
