TIME-RESOLVED POLARIZED FLUORESCENCE SPECTROSCOPY STUDIES OF PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 - CONFORMATIONAL-CHANGES OF THE REACTIVE CENTER UPON INTERACTIONS WITH TARGET PROTEASES, VITRONECTIN AND HEPARIN

Plasminogen activator inhibitor type 1 (PAI-1) is an important physiological inhibitor of the plasminogen activator system. To investigate the structure-functional aspects of this inhibitor, we have taken advantage of the lack of cysteine residues in the PAI-1 molecule and substituted Ser344 (P3) and Met347 (P1'), in the reactive center loop, with cysteines, thereby creating unique attachment sites for extrinsic fluorescent probe. Both cysteine mutants were purified and labeled with a sulfhydryl specific fluorophore, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacenyl-3-propionyl)-N-(iodoacetyl)ethylenediamine (BDYIA). The labeled mutants were found to reveal biochemical characteristics very similar to those of wild type PAI-1. Time-resolved fluorescence spectroscopy was used to examine orientational freedom of BDYIA in the reactive center loop of PAI-1. The orientational freedom of the probe was found to be greater in the latent form than in the active form of PAI-1, suggesting that the reactive center has a more relaxed conformation in the latent form than in the active form. Complex-formation with target proteases, tissue type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA), caused decreased orientational freedom of BDYIA in the P3 position, while the orientational freedom of BDYIA in position P1' increased to a level similar to that of BDYIA in reactive center-cleaved PAI-1. In contrast, complex formation with modified anhydro-uPA, which is unable to cleave its substrate, largely restricted the orientational freedom of BDYIA probe in the P1' position. Together, these findings suggest that the P1-P1' bond of the BDYIA-labeled PAI-I mutants is cleaved in the native complex with PAs. Since vitronectin and heparin interact with PAI-1, their influence on the orientational freedom of BDYIA in the reactive center of the PAI-1 molecule was also studied. The fluorescence anisotropy suggests that interactions with vitronectin and heparin induce conformational changes in the reactive center, indicating that PAI-1 has a mobile reactive center loop which is conformationally linked to both the vitronectin and heparin binding sites.