title: Additivity in effects of vitronectin and monoclonal antibodies against alpha-helix F of plasminogen activator inhibitor-1 on its reactions with target proteinases.
abstract: The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential therapeutic target in cardiovascular and cancerous diseases. PAI-1 circulates in blood as a complex with vitronectin. A PAI-1 variant (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-3-diazole (NBD) P9 PAI-1) with a fluorescent tag at the reactive center loop (RCL) was used to study the effects of vitronectin and monoclonal antibodies (mAbs) directed against alpha-helix F (Mab-2 and MA-55F4C12) on the reactions of PAI-1 with tissue-type and urokinase-type plasminogen activators. Both mAbs delay the RCL insertion and induce an increase in the stoichiometry of inhibition (SI) to 1.4-9.5. Binding of vitronectin to NBD P9 PAI-1 does not affect SI but results in a 2.0-6.5-fold decrease in the limiting rate constant (klim) of RCL insertion for urokinase-type plasminogen activator at pH 6.2-8.0 and for tissue-type plasminogen activator at pH 6.2. Binding of vitronectin to the complexes of NBD P9 PAI-1 with mAbs results in a decrease in klim and in a 1.5-22-fold increase in SI. Thus, vitronectin and mAbs demonstrated additivity in the effects on the reaction with target proteinases. The same step in the reaction mechanism remains limiting for the rate of RCL insertion in the absence and presence of Vn and mAbs. We hypothesize that vitronectin, bound to alpha-helix F on the side opposite to the epitopes of the mAbs, potentiates the mAb-induced delay in RCL insertion and the associated substrate behavior by selectively decreasing the rate constant for the inhibitory branch of PAI-1 reaction (ki). These results demonstrate that mAbs represent a valid approach for inactivation of vitronectin-bound PAI-1 in vivo.
authors.affiliation: Division of Biochemical Research, Department of Pathology, Henry Ford Health System, Detroit, Michigan 48202, USA. akomiss1@hfhs.org
authors.forename: Andrey A
authors.initials: AA
authors.lastname: Komissarov
authors.forename: Peter A
authors.initials: PA
authors.lastname: Andreasen
authors.forename: Julie S
authors.initials: JS
authors.lastname: Bødker
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Declerck
authors.forename: John Y
authors.initials: JY
authors.lastname: Anagli
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Shore
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000378:metabolism*; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D011480:Protease Inhibitors / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D013816:Thermodynamics; D010959:Tissue Plasminogen Activator / Q000378:metabolism; D017186:Titrimetry; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism; D019096:Vitronectin / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D000911:Antibodies, Monoclonal; D008024:Ligands; D017395:Plasminogen Activator Inhibitor 1; D011480:Protease Inhibitors; D019096:Vitronectin; D010447:Peptide Hydrolases; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator