Mechanisms of conversion of plasminogen activator inhibitor 1 from a suicide inhibitor to a substrate by monoclonal antibodies.
We have delineated two different reaction mechanisms of monoclonal antibodies (mAbs), MA-8H9D4 and either MA-55F4C12 or MA-33H1F7, that convert plasminogen activator inhibitor 1 (PAI-1) to a substrate for tissue (tPA)- and urokinase plasminogen activators. MA-8H9D4 almost completely (98-99%) shifts the reaction to the substrate pathway by preventing disordering of the proteinase active site. MA-8H9D4 does not affect the rate-limiting constants (k(lim)) for the insertion of the reactive center loop cleaved by tPA (3.5 s(-1)) but decreases k(lim) for urokinase plasminogen activator from 25 to 4.0 s(-1). MA-8H9D4 does not cause deacylation of preformed PAI-1/proteinase complexes and probably acts prior to the formation of the final inhibitory complex, interfering with displacement of the acylated serine from the proteinase active site. MA-55F4C12 and MA-33H1F7 (50-80% substrate reaction) do not interfere with initial PAI-1/proteinase complex formation but retard the inhibitory pathway by decreasing k(lim) (>10-fold for tPA). Interaction of two mAbs with the same molecule of PAI-1 has been directly demonstrated for pairs MA-8H9D4/MA-55F4C12 and MA-8H9D4/MA-33H1F7 but not for MA-55F4C12/MA-33H1F7. The strong functional additivity observed for MA-8H9D4 and MA-55F4C12 demonstrates that these mAbs interact independently and affect different steps of the PAI-1 reaction mechanism.
Komissarov
Andrey A
AA
Division of Biochemical Research, Henry Ford Health System, Detroit, Michigan 48202, USA.
Declerck
Paul J
PJ
Shore
Joseph D
JD
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007700:Kinetics; D051379:Mice; D008956:Models, Chemical; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism*; D011485:Protein Binding; D012694:Serine / Q000378:metabolism; D013347:Subcellular Fractions; D013379:Substrate Specificity; D010959:Tissue Plasminogen Activator / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D000911:Antibodies, Monoclonal; D017395:Plasminogen Activator Inhibitor 1; D012694:Serine; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator