Murine monoclonal antibodies against active site epitopes on human endothelial plasminogen activator inhibitor (PAI-1)

Summary A panel of 10 murine monoclonal antibodies was produced against human endothelial PAI-1 that had been purified to homogeneity in the absence of denaturants. All the antibodies bound to iodinated PAI-1 but no binding was observed to iodinated α 1 -antitrypsin (α 1 -AT), antithrombin III (ATIII) heparin cofactor II or α 2 -antiplasmin (α 2 -AP). PAI-1 was presented in the form of a complex with t-PA immobilised to microplate wells. Three of the 10 monoclonal antibodies (ESPI-1, ESPI-2 and ESPI-12) bound to the complexed PAI-1 while the others did not. Effects of the antibodies upon the interaction between PAI-1 and t-PA were measured by determining the distribution of 125 I-labelled t-PA between free and complexed forms after SDS-PAGE and autoradiography. Two clones (ESPI-4 and ESPI-6) reduced complex formation markedly when incubated with PAI-1 prior to its reaction with 125 I t-PA. ESPI-4 and ESPI-6 were also effective at quenching the activity of PAI-1 measured as reduction in t-PA activity in a chromogenic peptide substrate assay. Partial quenching of PAI-1 activity was achieved with ESPI-1 and ESPI-12. Thus 4 types of antibody reactivity towards PAI-1 could be distinguished. ESPI-3, 8, 10, 11 and 14 bound free PAI-1 (by ELISA or tracer binding assay) but did not bind complexed PAI-1 nor influence PAI-1 biological activity. ESPI-4 and 6 did not bind complexed PAI-1 but inhibited PAI-1 activity and prevented complex formation, ESPI-1 and 12 reacted with complexed PAI-1 and exhibited some inhibition of PAI-1 activity. Finally, ESPI-2 reacted with complexed PAI-1 but had no effect upon PAI-1 activity. The high affinity binding of PAI-1 by 3 of the antibodies immobilised on Sepharose 4B suggested their suitability for preparing PAI-1 depleted plasma and reagents. The other antibodies were effective as immunoaffinity purification reagents. They retained their capacity to bind PAI-1 over several cycles and the PAI-1 could be eluted with 0.1 M glycine-HC1 pH 3.0. The panel of antibodies will have a wide range of applications as assay, immunoaffinity purification and immunocytochemical reagents.