Metalloproteinase inhibitors inhibit the growth factor effect on pro-uPA activation and fibrinolytic activity. (A) Cell ELISA measurement of pro-uPA changes on the surface of VEGF-stimulated endothelial cells in the presence or absence of different protease inhibitors. 1,10-Phenanthroline (10 μM) as well as a specific gelatinase inhibitor (1 μM) prevented VEGF₁₆₅ (50 ng/mL)-induced pro-uPA reduction from the cell surface, while benzamidine (10 μM) or aprotinin (50 kIE/mL) had no effect. Results are given in percent of fluorescence units of unstimulated cells, whereby single bars represent average of mean values ± SDs of 3 independent experiments. V indicates VEGF₁₆₅; benz, benzamidine; apr, aprotinin; phe, 1,10-phenanthroline; M2/9I, ,MMP-2/9 inhibitor; M3I, MMP-3 inhibitor; and M8I, MMP-8 inhibitor. ^**P < .01 (n = 3). (B) d-dimer concentrations of supernatants from endothelial cells seeded on fibrin gels measured by a specific ELISA. Fresh supernatant was collected after 60 minutes. The VEGF₁₆₅ effect was diminished in the presence of a specific gelatinase inhibitor (M2/9I, 1 μM). Single bars represent mean values of 3 independent experiments; error bars represent SDs. (C) Western blots for MMP-2 and MMP-9 from VEGF₁₆₅-stimulated endothelial cell lysates. Specific mAbs against MMP-2 and MMP-9, respectively, were applied and visualized with subsequent chemiluminescence detection. Upper panel represents MMP-9 (zymogen 92 kDa and active form 82 kDa); lower panel, MMP-2 (zymogen 72 kDa and active form 62 kDa). (D) Gelatin zymography of endothelial cell lysates. The 62-kDa MMP-2 band significantly increases in intensity upon VEGF₁₆₅ stimulation.