title: Remarkable stabilization of plasminogen activator inhibitor 1 in a "molecular sandwich" complex.
abstract: Plasminogen activator inhibitor 1 (PAI-1) levels are elevated in a number of life-threatening conditions and often correlate with unfavorable outcomes. Spontaneous inactivation due to active to latent transition limits PAI-1 activity in vivo. While endogenous vitronectin (Vn) stabilizes PAI-1 by 1.5-2.0-fold, further stabilization occurs in a "molecular sandwich" complex (MSC) in which a ligand that restricts the exposed reactive center loop is bound to PAI-1/Vn. The effects of S195A two-chain urokinase (tcuPA) and Vn on inactivation of wild-type (wt) glycosylated (Gl-PAI-1), nonglycosylated (rPAI-1), and nonglycosylated Q123K PAI-1 (lacks Vn binding) forms were studied. S195A tcuPA decreased the rate constant (kL) for spontaneous inactivation at 37 °C for rPAI-1, Q123K, and Gl-PAI-1 by 6.7-, 3.4-, and 7.8-fold, respectively, and both S195A tcuPA and Vn by 66.7-, 5.5-, and 103.3-fold, respectively. Analysis of the temperature dependences of kL revealed a synergistic increase in the Gibbs free activation energy for spontaneous inactivation of wt Gl-PAI-1 and rPAI-1 in MSC from 99.8 and 96.1 to 111.3 and 107.0 kJ/mol, respectively, due to an increase in the activation enthalpy and a decrease in the activation entropy. Anti-PAI-1 monoclonal antibodies (mAbs) competing with proteinase also stabilize PAI-1/Vn. The rate of inhibition of target proteinases by MSCs, with a stoichiometry close to unity, was limited by the dissociation (k = 10(-4) to 10(-3) s(-1)) of S195A tcuPA or mAb. The stabilization of PAI-1 in MSCs in vivo may potentiate uncontrolled thrombosis or extravascular fibrin deposition, suggesting a new paradigm for using PAI-1 inhibitors and novel potential targets for therapy.
authors.affiliation: Texas Lung Injury Institute, University of Texas Health Science Center at Tyler , 11937 U.S. Highway 271, Tyler, Texas 75708-3154, United States.
authors.forename: Galina
authors.initials: G
authors.lastname: Florova
authors.forename: Sophia
authors.initials: S
authors.lastname: Karandashova
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Declerck
authors.forename: Steven
authors.initials: S
authors.lastname: Idell
authors.forename: Andrey A
authors.initials: AA
authors.lastname: Komissarov
mesh_terms: D000577:Amides / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D007700:Kinetics; D008958:Models, Molecular; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000276:immunology; D055550:Protein Stability; D013816:Thermodynamics
chemical_list: D000577:Amides; D000911:Antibodies, Monoclonal; D017395:Plasminogen Activator Inhibitor 1