Functional characterization of monoclonal antibodies directed against fibrin binding domains of tissue-type plasminogen activator.
Fibrin interacts with tissue-type plasminogen activator (tPA) via the finger and the kringle 2 domains. Three monoclonal antibodies against tPA, designated MPW3VPA, MPW6VPA, and MPW7VPA, which react with epitopes in the tPA molecule involved in fibrin binding, were characterized. The IgM monoclonal antibody MPW6VPA, directed against an epitope close to the finger and epidermal growth factor domains, stimulated plasminogen activation only in the absence of CNBr-fibrinogen fragments by increasing kcat in a dose-dependent fashion, an effect which was not restricted to the intact molecule. These results suggest that MPW6VPA mimics the initial effect of fibrin bound to the tPA molecule, which results in a change of kcat values. The MPW6VPA effect was reversed by another antibody, MPW3VPA, also directed against epidermal growth factor and finger domains. The latter antibody also inhibited plasminogen activation by tPA in the presence of CNBr-fibrinogen fragments in a dose-dependent, apparently noncompetitive way. No effect of MPW3VPA was seen in the absence of CNBr-fibrinogen fragments. MPW7VPA directed against kringle 2 of tPA inhibited plasminogen activation by tPA only when CNBr-fibrinogen fragments were present. This inhibition was apparently competitive and dose-dependent. These data suggest that MPW3VPA interferes with the first phase of fibrin binding to tPA, whereas MPW7VPA interferes with the second phase of fibrin binding to the tPA molecule via kringle 2, resulting in Km changes.
Wojta
J
J
Department of Medical Physiology, University of Vienna, Austria.
Beckmann
R
R
Turcu
L
L
Wagner
O F
OF
van Zonneveld
A J
AJ
Binder
B R
BR
D000911:Antibodies, Monoclonal / Q000276:immunology*; D001665:Binding Sites; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000378:metabolism; D000939:Epitopes / Q000276:immunology; D005337:Fibrin / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism / Q000494:pharmacology; D007075:Immunoglobulin M / Q000276:immunology; D007700:Kinetics; D008970:Molecular Weight; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D010958:Plasminogen / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007075:Immunoglobulin M; D010446:Peptide Fragments; D004815:Epidermal Growth Factor; D005337:Fibrin; D005340:Fibrinogen; D010958:Plasminogen; D010959:Tissue Plasminogen Activator; D003488:Cyanogen Bromide