A regulatory hydrophobic area in the flexible joint region of plasminogen activator inhibitor-1, defined with fluorescent activity-neutralizing ligands - Ligand-induced serpin polymerization

We have characterized the neutralization of the inhibitory activity of the serpin plasminogen activator inhibitor-1 (PAI-1) by a number of structurall y distinct organo-chemicals, including compounds with environment-sensitive spectroscopic properties. In contrast to latent and reactive center-cleave d PAI-1 and PAI-1 in complex with urokinase-type plasminogen activator (uPA ), active PAI-1 strongly increased the fluorescence of the PAI-1-neutralizi ng compounds 1-anilinonaphthalene-8-sulfonic acid and 4,4'-dianilino-1,1'-b isnaphthyl-5,5'-dis acid, The fluorescence increase could be competed by al l tested nonfluorescent neutralizers, indicating that all neutralizers bind to a common hydrophobic area preferentially accessible in active PAI-1, Ac tivity neutralization proceeded through two consecutive steps as follows: f irst step is conversion to forms displaying substrate behavior toward uPA, and second step is to forms inert to uPA With some neutralizers, the second step was associated with PAI-1 polymerization, Vitronectin reduced the sus ceptibility to the neutralizers. Changes in sensitivity to activity neutral ization by point mutations were compatible with the various neutralizers ha ving overlapping but not identical, binding sites in the region around alph a -helices D and E and beta -strand 1A, known to act as a flexible joint wh en beta -sheet A opens and the reactive center loop inserts as beta -strand 4A during reaction with target proteinases. The defined binding area may b e a target for development of compounds for neutralizing PAI-1 in cancer an d cardiovascular diseases.