The Mechanism of Binding of Low-Molecular-Weight Active Site Inhibitors to Human α-Thrombin

Abstract

The thrombin inhibitors argatroban, efegatran, NAPAP, CH 1091, CH 248, inogatran and melagatran have been characterised with respect to their mechanism of binding to human α-thrombin. Stopped-flow spectrophotometry was used to follow thrombin-catalysed hydrolysis of the chromogenic substrate S-2238 in the presence of inhibitors. The rate of onset or decay of inhibition was evaluated using progress curve analysis. It was possible to obtain apparent association and dissociation rate constants from the dependence of the rates on the inhibitor concentrations. Inhibition constants calculated from the association and dissociation rate constants were in good agreement with those calculated from steady-state rates. The binding of 6 inhibitors was also monitored directly using stopped-flow spectrofluorimetry when two kinetic components were found with all inhibitors. The faster component accounted for the largest part of the change in the intrinsic fluorescence of thrombin induced by inhibitor binding and was dependent on the inhibitor concentration. The slower component was independent of the concentration of the inhibitor. The concentration dependence of the faster component was linear with the compounds argatroban, NAPAP, CH 1091 and melagatran and hyperbolic with the compounds CH 248 and inogatran. The values of the apparent second-order rate constants at pH 7.4 and 37°C range from slow to rapid binding in the interval 16–78 × 10⁶M^-1s^-1, which is somewhat higher than 1–34 × 10⁶M^-1s^-1 obtained from progress curve analysis of the onset of inhibition.

The present results support a mechanism that includes rearrangement of a weak initial thrombin-inhibitor complex towards a tighter complex. Moreover, at least one additional step is required in the mechanism. In this model, the rate-limiting step for the binding of the inhibitor at concentrations in the nanomolar range depends on the primary interaction between the inhibitor and native thrombin.