title: Mechanism of inactivation of plasminogen activator inhibitor-1 by a small molecule inhibitor.
abstract: The inactivation of plasminogen activator inhibitor-1 (PAI-1) by the small molecule PAI-1 inhibitor PAI-039 (tiplaxtinin) has been investigated using enzymatic analysis, direct binding studies, site-directed mutagenesis, and molecular modeling studies. Previously PAI-039 has been shown to exhibit in vivo activity in various animal models, but the mechanism of inhibition is unknown. PAI-039 bound specifically to the active conformation of PAI-1 and exhibited reversible inactivation of PAI-1 in vitro. SDS-PAGE indicated that PAI-039 inactivated PAI-1 predominantly through induction of PAI-1 substrate behavior. Preincubation of PAI-1 with vitronectin, but not bovine serum albumin, blocked PAI-039 activity while analysis of the reciprocal experiment demonstrated that preincubation of PAI-1 with PAI-039 blocked the binding of PAI-1 to vitronectin. Together, these data suggest that the site of interaction of the drug on PAI-1 is inaccessible when PAI-1 is bound to vitronectin and may overlap with the PAI-1 vitronectin binding domain. This was confirmed by site-directed mutagenesis and molecular modeling studies, which suggest that the binding epitope for PAI-039 is localized adjacent to the previously identified interaction site for vitronectin. Thus, these studies provide a detailed characterization of the mechanism of inhibition of PAI-1 by PAI-039 against free, but not vitronectin-bound PAI-1, suggesting for the first time a novel pool of PAI-1 exists that is vulnerable to inhibition by inactivators that bind at the vitronectin binding site.
authors.affiliation: Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109-0644, USA.
authors.forename: Natalia V
authors.initials: NV
authors.lastname: Gorlatova
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: Cale
authors.forename: Hassan
authors.initials: H
authors.lastname: Elokdah
authors.forename: Donghua
authors.initials: D
authors.lastname: Li
authors.forename: Kristi
authors.initials: K
authors.lastname: Fan
authors.forename: Mark
authors.initials: M
authors.lastname: Warnock
authors.forename: David L
authors.initials: DL
authors.lastname: Crandall
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Lawrence
mesh_terms: D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D006031:Glycosylation; D006801:Humans; D007210:Indoleacetic Acids / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance; D013997:Time Factors; D019096:Vitronectin / Q000737:chemistry
chemical_list: D007210:Indoleacetic Acids; D017395:Plasminogen Activator Inhibitor 1; D019096:Vitronectin; C488103:tiplaxtinin