Mechanism of the inhibition of α‐thrombin by hirudin‐derived fragments hirudin(1–47) and hirudin(45–65)

Abstract

The kinetic mechanism of the inhibition of α‐thrombin by hirudin was analyzed using the hirudin‐derived fragments hirudin(1–47) and hirudin(45–65). Previously, these fragments have been shown to interact with α‐thrombin at distinct sites inhibiting thrombin‐mediated clot formation. Binding to the active site the N‐terminal fragment hirudin (1–47) competitively inhibits hydrolysis of the substrates Tos‐Gly‐Pro‐Arg‐NH‐Mec (Tos, tosyl; NH‐Mec, 4‐methylcoumaryl‐7‐amide) and fibrinogen with Ki values of 420 ± 18 nM and 460 ± 25 nM, respectively. Interacting with the anion‐binding site of α‐thrombin the C‐terminal fragment competitively inhibits the hydrolysis of fibrinogen with a Ki of 760 ± 40 nM. It was found, however, that this fragment acts as a hyperbolic uncompetitive inhibitor with respect to the hydrolysis of the peptide‐NH‐Mec substrate. According to the Botts‐Morales scheme for enzyme inhibition, the parameters Ki= 710 ± 38 nM, K′i= 348 ± 22 nM, as well as α=β= 0.49 of thrombin inhibition by the C‐terminal fragment hirudin(45–65), were obtained. The results are discussed in terms of the interaction of hirudin and thrombin. Copyright © 1991, Wiley Blackwell. All rights reserved