Characterization of human tissue-type plasminogen activator with monoclonal antibodies: mapping of epitopes and binding sites for fibrin and lysine.
The study defines interactions between human tissue-type plasminogen activator (t-PA) and 21 mouse monoclonal antibodies (mAb). Characterization includes epitope distribution, reactivity of different forms of t-PA with antibodies, and modification of t-PA function by antibody binding. Eighteen antibodies are directed against t-PA A-chain. These antibodies recognize four distinct epitopes (A, B, C, D) and one partially overlapping epitope (D'). The remaining three antibodies are directed against two different epitopes (E, F) on catalytically active t-PA B-chain. A-chain reactive antibodies do not bind to the reduced form of t-PA, while B-chain reactive antibodies bind to reduced and deglycosylated t-PA forms. The latter antibodies associate more tightly with sc t-PA than with tc t-PA and have a higher affinity for t-PA-PAI 1 complex as compared to free t-PA. The analysis of functional effects of antibodies reveals that antibodies directed against all above defined epitopes inhibit interactions between t-PA and fibrin: a) binding of t-PA to fibrin, b) fibrinolytic activity of t-PA, and c) fibrin activation of sc t-PA amidolytic activity. The observations support the assumption that several sites of t-PA are involved in fibrin binding and that fibrin-bound t-PA is closely surrounded by the fibrin mesh. Many antibodies quench also binding of t-PA to lysine-Sepharose. Experiments with free, non-fixed lysine confirm strong competition between lysine and mAb 16 and 18, directed against epitope A, and mAb 29, binding to epitope F.(ABSTRACT TRUNCATED AT 250 WORDS)
Zacharias
U
U
Central Institute of Molecular Biology, Berlin-Buch, FRG.
Fischer
B
B
Noll
F
F
Will
H
H
D000911:Antibodies, Monoclonal; D001665:Binding Sites; D000939:Epitopes / Q000737:chemistry; D005337:Fibrin / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D007120:Immunochemistry; D007124:Immunoenzyme Techniques; D008024:Ligands; D008239:Lysine / Q000737:chemistry; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D008024:Ligands; D005337:Fibrin; D010959:Tissue Plasminogen Activator; D008239:Lysine