Identification of the binding site of Glu-plasminogen to β2-GPI by inhibition ELISA using plasminogen fragments. (A) Binding of Glu-plasminogen to immobilized nicked β2-GPI was tested by ELISA in the presence of possible inhibitors. After nicked β2-GPI immobilization onto microtiter plates, different concentrations of kringle 4 of plasminogen (○) or mini-plasminogen (that consists of kringle 5 and catalytic domain of plasminogen; •) were added as inhibitors. BSA (▪) served as control. After incubation and washing, Glu-plasminogen (10 μg/mL) was added and bound Glu-plasminogen was determined using kringle 1- to 3-specific mouse monoclonal antiplasminogen antibody. (B) For the inhibition ELISA kringle 1 to 3 of plasminogen (○) or mini-plasminogen (•) served as inhibitors. Glu-plasminogen bound to immobilized β2-GPI was detected using kringle 4-specific mouse monoclonal antiplasminogen antibody. Assays were run in triplicate. (C) Competitive ELISA using EACA, a lysine homologue. Binding of nicked β2-GPI (0.2 μM) to immobilized Glu-plasminogen was tested by ELISA using Cof-22 antibody in the presence of various concentrations of EACA (0-0.20 μg/mL). (D) Soluble fibrin monomer (5 μg/mL) was coated on the surface of a microtiter plate and blocked. Biotinylated Glu-plasminogen (5 μg/mL) was preincubated with intact or nicked β2-GPI and added to the wells. After incubation and washing, ALP-conjugated streptavidin was used for detection. Assays were run triplicate. Error bars indicate SDs. K indicates kringle; mini-plg, mini-plasminogen.
