THE MECHANISM OF BINDING OF LOW-MOLECULAR-WEIGHT ACTIVE-SITE INHIBITORS TO HUMAN ALPHA-THROMBIN

The thrombin inhibitors argatroban, efegatran, NAPAP, CH 1091, CH 248, inogatran and melagatran have been characterised with respect to thei r mechanism of binding to human alpha-thrombin. Stopped-flow spectroph otometry was used to follow thrombin-catalysed hydrolysis of the chrom ogenic substrate S-2238 in the presence of inhibitors. The rate of ons et 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 concentrat ions. Inhibition constants calculated from the association and dissoci ation rate constants were in good agreement with those calculated from steady-slate rates. The binding of 6 inhibitors was also monitored di rectly using stopped-flow spectrofluorimetry when two kinetic componen ts were found with all inhibitors. The faster component accounted for the largest part of the change in the intrinsic fluorescence of thromb in induced by inhibitor binding and was dependent on the inhibitor con centration. 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 melagatr an and hyperbolic with the compounds CH 248 and inogatran. The values of the apparent second-order rate constants at pH 7.4 and 37 degrees C range from slow to rapid binding in the interval 16-78 x 10(6) M-1 s( -1), which is somewhat higher than 1-34 x 10(6) M-1 s(-1) obtained fro m progress curve analysis of the onset of inhibition. The present resu lts support a mechanism that includes rearrangement of a weak initial thrombin-inhibitor complex towards a tighter complex. Moreover, at lea st one additional step is required in the mechanism. In this model, th e rate-limiting step for the binding of the inhibitor al concentration s in the nanomolar range depends on the primary interaction between th e inhibitor and native thrombin.