Citation

Komissarov AA, Declerck PJ, Shore JD. J. Biol. Chem. 2002; 277(46): 43858-43865.

Copyright

(Copyright © 2002, American Society for Biochemistry and Molecular Biology)

DOI

10.1074/jbc.M204110200

PMID

12223472

Abstract

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.

Language: en

Keywords

Animals; Antibodies, Monoclonal; Binding Sites; Chromatography, Gel; Dose-Response Relationship, Drug; Humans; Kinetics; Mice; Models, Chemical; Plasminogen Activator Inhibitor 1; Protein Binding; Serine; Subcellular Fractions; Substrate Specificity; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator