Kinetic studies are presented which show that native human vitronectin, but not urea-treated vitronectin, accelerates the inactivation of human alpha-thrombin by human plasminogen activator inhibitor-1 (PAI-1). We demonstrate that although urea-treated vitronectin binds PAI-1 with an affinity greater than that of native vitronectin, it does not accelerate the rate of inactivation of alpha-thrombin by PAI-1. We present evidence to suggest that the inability of urea-treated vitronectin to accelerate the reaction between alpha-thrombin and PAI-1 results at least in part from the inability of urea-treated vitronectin to bind to alpha-thrombin. The accelerated reaction between PAI-1 and alpha-thrombin can be accounted for by the formation of a tight complex between native vitronectin and PAI-1 that reacts in a saturable manner (Kd = 75 nM) with alpha-thrombin. The second-order rate constant (kI/Kd) 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 (610 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 vitronectin-PAI-1 complex, as reflected by a greater than 25-fold reduction in the dissociation constant (Kd) observed for the vitronectin-PAI-1 complex relative to that of uncomplexed PAI-1.