cr-Thrombin-catalyzed Hydrolysis of Fibrin I ALTERNATIVE BINDING MODES AND THE ACCESSIBILITY OF THE ACTIVE SITE IN FIBRIN I-BOUND a-THROMBIN*

Steady-state kinetic parameters were determined for the action of human a-thrombin on human fibrin I polymer, an intermediate in the a-thrombin-catalyzed conversion of fibrinogen to the fibrin matrix of blood clots during the terminal phase of the blood clotting cascade. Values of 49 s-’ and 7.5 pM were determined (at 37 “C, pH 7.4, I’/2 0.17) for Lt and K,, respectively. Studies of the effect of fibrin I on a-thrombincatalyzed hydrolysis of the fluorogenic substrate Np-Tos-Gly-L-Pro-L-Arg-7-amido-4-methylcoumarin (tos-GPR-amc) and the effect of fibrin I on the reaction of cy-thrombin with antithrombin III (AT) were presented which indicate that the active site of a-thrombin is accessible while it is bound to its substrate fibrin I. Fibrin I inhibited cY-thrombin-catalyzed hydrolysis of tos-GPR-amc in a manner inconsistent with the pure competitive inhibition expected for an alternative substrate, whereas fibrinogen, an a-thrombin substrate, behaved as a pure competitive inhibitor of the a-thrombin-catalyzed hydrolysis of tos-GPR-amt. The effect of fibrin I on a-thrombin-catalyzed hydrolysis of tosGPR-amc was shown to be consistent with a-thrombin binding to fibrin I in alternative orientations. In one orientation both the active site and a site distinct from the active site (an exosite) of a-thrombin are occupied by fibrin I. In the other orientation only the exosite of a-thrombin is occupied and the active site is freely accessible to other substrates. The values of both k,,, (21 s-l) and K, (co.23 FM) determined for fibrin Ibound a-thrombin acting on tos-GPR-amc were decreased relative to the values of kcat (180 s-l) and K, (7.3 pM) observed for the action of uncomplexed athrombin on tos-GPR-amt. This observation suggests that the active site of a-thrombin is altered in fibrin Ibound a-thrombin. Studies of the effect of fibrin I on the reaction of AT with a-thrombin (at 37 “C, pH 7.4, l?/2 0.17) indicated that when a-thrombin is bound to fibrin I in an orientation where the active site of athrombin is accessible, AT reacts with a-thrombin with a rate constant (>4.2 % lo4 M-’ s-l) that is greater than the rate constant (1.5 x lo4 MS1 s-‘) for reaction of AT with the free enzyme. It is likely that the ability of fibrin I to bind a-thrombin in an orientation where the active site of a-thrombin is accessible is an important determinant of the plasma lifetime of active a-throm-