A MONOCLONAL-ANTIBODY DIRECTED AGAINST AN EPITOPE IN THE NH2-TERMINALREGION OF NATIVE HUMAN PLASMINOGEN INDUCES A MODIFICATION OF ITS FUNCTIONAL-PROPERTIES

A monoclonal antibody (MAb A1D12) that is directed against an epitope localized in the amino acid sequence Arg(68)-Lys(77) of human plasmino gen was identified. Binding of MAb A1D12 to native plasminogen resulte d in a 2- to 3-fold enhanced plasminogen activation by various plasmin ogen activators and an up to 2-fold enhanced binding to fibrin(ogen). Consequently, MAb A1D12 potentiated the fibrinolysis in human plasma i nduced by single-chain urokinase-type plasminogen activator (scu-PA), two-chain urokinase-type plasminogen activator (tcu-PA), streptokinase (SK) and tissue-type plasminogen activator (t-PA), each to a differen t degree. Incorporation of both MAb A1D12 (at 200 mu g/ml) and the pla sminogen activator in human plasma before clotting resulted in enhance d subsequent lysis as compared to the absence of MAb: release of fibri n D-dimer after incubation for 15 min at 37 degrees C was enhanced 50- fold for tcu-PA, 15 to 20-fold for SK and only 3-fold for t-PA. The ge neration of fibrin D-dimer from whole blood clots submerged for 100 mi n at 37 degrees C in normal human plasma containing 150 mu g/ml of MA6 A1D12 was enhanced 2 to 3-fold, e-fold or 1.5-fold (as compared to th e absence of MAb) for scu-PA, tcu-PA, and SK, respectively. Fibrin D d imer levels were not significantly increased for t-PA. The differentia l effects of MAb A1D12 on the fibrinolytic potential of scu-PA, tcu-PA , SK or t-PA may be related to the different mechanism of action of th ese plasminogen activators. The effects of MAb A1D12 on the functional properties of plasminogen may be due to a conformational change from a tight spiral structure in native plasminogen to a more open structur e. Thereby, an intramolecular interaction between the NH2-terminal pre -activation peptide and the kringle and/or proteinase domains is disru pted, resulting in a better accessibility of the Arg(561)-Val(562) pep tide bond for plasminogen activators and of the lysine-binding sites i n the kringle domains for binding to fibrin. These data thus confirm t he functional importance of this intramolecular interaction in plasmin ogen and establish that it involves the region comprising amino acids Arg(68) through Lys(77).