KINETICS OF INACTIVATION OF ALPHA-THROMBIN BY PLASMINOGEN-ACTIVATOR INHIBITOR-1 - COMPARISON OF THE EFFECTS OF NATIVE AND UREA-TREATED FORMS OF VITRONECTIN

Kinetic studies are presented which show that native human vitronectin , but not urea-treated vitronectin, accelerates the inactivation of hu man alpha-thrombin by human plasminogen activator inhibitor-1 (PAI-1). We demonstrate that although urea-treated vitronectin binds PAI-1 wit h an affinity greater than that of native vitronectin, it does not acc elerate the rate of inactivation of alpha-thrombin by PAI-1. We presen t evidence to suggest that the inability of urea-treated vitronectin t o accelerate the reaction between alpha-thrombin and PAI-1 results at least in part from the inability of urea-treated vitronectin to bind t o alpha-thrombin. The accelerated reaction between PAI-1 and alpha-thr ombin can be accounted for by the formation of a tight complex between native vitronectin and PAI-1 that reacts in a saturable manner (K(d) = 75 nM) with alpha-thrombin. The second-order rate constant (k(I)/K(d )) for the reaction of the native vitronectin-PAI-1 complex with alpha -thrombin (1.64 x 10(5) M--1 s-1) is 270-fold greater than the second- order rate constant for the reaction in the absence of vitronectin (61 0 M-1 s-1). The increase in the second-order rate constant is largely due to an increase in the affinity of alpha-thrombin for the native vi tronectin-PAI-1 complex, as reflected by a greater than 25-fold reduct ion in the dissociation constant (K(d)) observed for the vitronectin-P AI-1 complex relative to that of uncomplexed PAI-1.