Potentiation of plasminogen activation by an anti-urokinase monoclonal antibody due to ternary complex formation. A mechanistic model for receptor-mediated plasminogen activation.
We have observed that a murine IgG1 monoclonal antibody directed against human urokinase-type plasminogen activator (uPA) greatly potentiates pro-uPA-mediated plasminogen activation. This effect was dependent on the interaction between the immunoglobulin and the kringle domain of pro-uPA and could be competed efficiently by kringle-containing proteolytic fragments of uPA. In addition, the potentiation could also be competed by the lysine analog 6-aminohexanoic acid, an antagonist of plasminogen binding. This unexpected plasminogen binding dependence was found to be due to a carboxyl-terminal lysine residue on the immunoglobulin gamma chain, which by analogy with other proteins represents a potential binding site for plasminogen. Removal of this residue with carboxypeptidase B resulted in a complete abolition of the potentiation. It appears therefore that the potentiatory effect involves a novel mechanism with the antibody acting to provide a specific template for the assembly of a ternary complex involving pro-uPA/uPA and plasminogen, enabling them to interact in a catalytically favorable manner. This interpretation was confirmed by studying the kinetics of plasminogen activation by the complex between active, two-chain uPA and the antibody, which resulted in an overall 50-fold increase in reaction efficiency (kcat/Km), primarily due to a reduction in Km from 20 to 0.1 microM. Pro-uPA activation by plasmin was also accelerated, although to a lesser extent. The potentiation due to complex formation also provides a mechanism for the initiation of this system, dependent only on the low intrinsic proteolytic activity of the zymogen forms. The effects observed here, mediated by ternary complex formation, simulate the effects we have previously observed on assembly of the uPA receptor-mediated cellular plasminogen activation system and may therefore represent a mechanistic model for both its activity and initiation.
Ellis
V
V
Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark.
Danø
K
K
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D000936:Antigen-Antibody Complex; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D010958:Plasminogen / Q000378:metabolism*; D011487:Protein Conformation; D011498:Protein Precursors / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D011498:Protein Precursors; D010958:Plasminogen; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator