A monoclonal antibody directed against the high-affinity lysine-binding site (LBS) of human plasminogen. Role of LBS in the regulation of fibrinolysis

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Abstract
One of thirty murine monoclonal antibodies, raised by immunization with human plasmin‐α2‐antiplasmin complex, was found to be directed against the high‐affinity lysine‐binding site in plasminogen. Indeed, this antibody (MA‐HAL) reacted with plasminogen and with a fragment of plasminogen composed of the first three triple‐loop structures (LBS I) and was displaced by 6‐aminohexanoic acid (50% displacement at 25 μM). In competitive radioimmunoassays the binding of radiolabeled plasminogen to MA‐HAL was reduced to 50% with 2.3 μM α2‐antiplasmin or 1.3 μM histidine‐rich glycoprotein, which corresponds to the known dissociation constants between these ligands and the high‐affinity lysine‐binding site of plasminogen. MA‐HAL did not influence the activation of plasminogen by tissue‐type plasminogen activator in the absence of CNBr‐digested fibrinogen, but abolished the effect of CNBr‐digested fibrinogen on the Michaelis constant of the reaction. MA‐HAL reduced the reaction rate between plasmin and α2‐antiplasmin by a factor 20 and abolished the binding of plasminogen to fibrin. These results indicate that MA‐HAL specifically binds to and masks the high‐affinity lysine‐binding site of plasminogen. It therefore is a useful tool for the investigation of the role of this structure in the regulation of fibrinolysis, both at the level of fibrin‐stimulated activation of plasminogen and of the inhibition of generated plasmin.