In a recent study it was demonstrated that thrombin des-PPW reacts with antithrombin (AT) very poorly. In this study it is shown that a Trp to Ala (W60A) mutant of thrombin also reacts with AT at a lower rate than thrombin. The inhibition kinetics were studied by the slow-binding kinetic approach. In both the presence and absence of heparin, the pseudo-first-order rate constant of thrombin inhibition (kobs) increased linearly with AT concentration, indicating that inhibition, in the concentration range covered, conforms to a bimolecular reaction E+I-->K assn E-I. Only the second-order association rate constant (kassn) for thrombin can be estimated [6.8 +/- 2.7) x 10(3) M-1 s-1 in the absence of heparin and (4.1 +/- 1.2) x 10(6) M-1 s-1 in the presence of heparin]. With W60A and des-PPW, the kobs of inhibition increased hyperbolically as a function of AT concentration, indicating that the inhibition is a two-step process according to E+I<==>K init E.I-->k2 E-I. The kinetic constants for W60A were estimated to be Kinit = 13.6 +/- 3.3 microM and k2 = 0.007 +/- 0.001 s-1 in the absence of heparin and K init = 13.6 +/- 3.1 nM and k2 = 0.008 +/- 0.002 s-1 in the presence of heparin. AT inhibited des-PPW very slowly [K assn = (2.9 +/- 0.7) x 10(1) M-1 s-1], but heparin accelerated the reaction approximately 20,000-fold and made it possible to demonstrate a two-step reaction mechanism for des-PPW with K init = 10.4 +/- 2.3 nM and k2 = 0.006 +/- 0.001 s-1. In contrast to thrombin, an active AT-binding pentasaccharide enhanced the inhibition of des-PPW approximately 15-fold. These results indicate that (1) in contrast to thrombin, the heparin-induced conformational change in AT is required for optimal inhibition of des-PPW and (1) Trp60 is essential for normal thrombin-AT reaction. On the basis of these results, a modified model for thrombin-AT interaction is proposed.