Kinetics of inactivation of alpha-thrombin by plasminogen activator inhibitor-1. Comparison of the effects of native and urea-treated forms of vitronectin

• Published in: The Journal of biological chemistry Vol. 268; no. 17; pp. 12367 - 12372
• Main Authors: NASKI, M. C, LAWRENCE, D. A, MOSHER, D. F, PODOR, T. J, GINSBURG, D
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 15.06.1993
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Blood Proteins - chemistry; Blood Proteins - pharmacology; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Glycoproteins - chemistry; Glycoproteins - metabolism; Glycoproteins - pharmacology; Humans; Hydrolases; Kinetics; Mathematics; Models, Theoretical; Plasminogen Activator Inhibitor 1 - metabolism; Plasminogen Activator Inhibitor 1 - pharmacology; Protein Binding; Protein Denaturation; Recombinant Proteins - metabolism; Recombinant Proteins - pharmacology; Thrombin - antagonists & inhibitors; Urea - pharmacology; Vitronectin; Human; Enzyme; Enzyme inhibitor; Molecular interaction; Plasminogen activator; Thrombin; Vitronectin; Kinetics; Inactivation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)31399-1

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.