Plasminogen is activated at the surface of EMPs by uPA. (A,B) A variable number of EMPs immobilized on poly-L-lysine surfaces were incubated with fixed concentrations of plasminogen (0.5 μM) and the plasmin-selective chromogenic substrate (0.75 mM). The graph in panel A shows an increase in the formation of plasmin as a function of the number of immobilized microparticles. Unbound reagents were then washed off and the chromogenic substrate added to detect plasmin that remained bound to the immobilized EMPs; the graph (B) shows an increase in bound plasmin as a function of the number of immobilized EMPs. Representative graphs (mean ± SD) of 3 independent experiments. (C,D) Protein extracts from HMEC-1 and its derived EMPs were processed for zymography (panel C) to detect plasminogen activator activity (5 μg protein/lane; S: EMP last washing supernatant) and for immunoblot (panel D) using rabbit antibodies against uPAR and purified uPAR as reference (20 μg protein/lane). The ∼50 kDa and upper bands in panel C were inhibited by antibodies to urokinase (not shown). A space has been inserted to indicate where a gel lane was cut. These gels came from different experiments as indicated by the space between the gels.