Summary
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. Weak
inhibition is exerted on association of mAb 2, 21, and 25 to epitope D. Amidolytic
activity is suppressed only by B-chain specific antibody 22.
