Identification of uPA, uPAR, and uPA/uPAR complex on EMP. (A,B) Measured fluorescence intensity by flow cytometry analysis of EMPs with anti-uPA (A) and anti-uPAR (B) antibodies (black) and the corresponding isotype controls (gray). (C) Representative images of immunogold labeling of uPA (top) and uPAR (bottom) analysis on EMPs by transmission electron microscopy. Slides were viewed with a JEOL 1220 transmission electron microscope (JEOL, Tokyo, Japan). Images were acquired using a MegaView III camera (Soft Imaging System, Munster, Germany) and were processed with Analysis software (Soft Imaging System) and Adobe Photoshop version 7.0 software (Adobe Systems, San Jose, CA). The dimension of the bars indicate the relative small size of these EMPs, and the clusters of 15-nm gold particles (black dots) indicate the presence of uPA and uPAR at the surface of EMPs. (D) Main graph: isotherm of the binding of varying amounts of scuPA incubated with EMPs immobilized on a poly-L-lysine surface. The amount of bound scuPA was detected by its ability to activate plasminogen using a chromogenic substrate selective for plasmin. Data fitted to the Langmuir equation as indicated in “Materials and methods” allowed calculation of a dissociation constant, Kd = 0.1 nM, for the interaction of scuPA with its receptor. Representative graph (mean ± SD) of 3 independent experiments. Inset: specificity of the binding of native scuPA is demonstrated by its inhibition with a modified recombinant form of scuPA (r-scuPA, ILe159→Gly) that bind to its receptor but cannot be activated. The bars represent the mean (± SD) of 3 independent experiments.