Characterization of functional domains in human tissue‐type plasminogen activator with the use of monoclonal antibodies

Abstract

Two murine monoclonal antibodies (MA‐2G6 and MA‐1C8), secreted by hybridomas obtained by fusion of myeloma cells with spleen cells from mice immunized with human tissue‐type plasminogen activator (t‐PA), inhibited the activity of t‐PA on fibrin plates. MA‐2G6 inhibited the amidolytic activity of t‐PA and did not react with t‐PA in which the active‐site serine was blocked with diisopropylfluorophosphate nor with t‐PA in which the active‐site histidine was alkylated by reaction with d‐Ile‐Pro‐Arg‐CH₂Cl. This indicated that MA‐2G6 is directed against an epitope covering the active site of t‐PA. MA‐1C8 did not inhibit the amidolytic activity of t‐PA, but abolished both the binding of t‐PA to fibrin and the stimulatory effect of fibrin on the activation of plasminogen by t‐PA. Thus MA‐1C8 is directed against an epitope which covers the fibrin‐binding site of t‐PA. The A and B chains of partially reduced two‐chain t‐PA were separated by immunoadsorption on immobilized MA‐1C8 and MA‐2G6. The purified B chain reacted with MA‐2G6 but not with MA‐1C8 and activated plasminogen following Michaelis‐Menten kinetics with kinetic constants similar to those of intact t‐PA (K_m= 100 μM and k_cat= 0.02 s⁻¹). However, fibrin or CNBr‐digested fibrinogen did not stimulate the activation of plasminogen by the B chain. The purified A chain reacted with MA‐1C8 but not with MA‐2G6. It bound to fibrin with an affinity similar to that of intact t‐PA but did not activate plasminogen. It is concluded that the active center of t‐PA is located in the B chain and the fibrin‐binding site in the A‐chain. Both functional domains are required for the regulation by fibrin of the t‐PA‐mediated activation of plasminogen.