VEGF stimulation of endothelial cells leads to pro-uPA activation on the cell surface. (A) Characterization of the anti-pro-uPA antibody PUK. Pro-uPA antibody reactivity versus uPA activity on the synthetic chromogenic substrate S-2444 was blotted in a standard curve obtained with different pro-uPA/uPA ratios in a solution of 10 ng/mL urokinase antigen. These data indicate that only the inactive pro-uPA is recognized by the PUK antibody, while active uPA is not. (B) VEGF₁₆₅ (50 ng/mL) induced cell surface uPA (dark gray bars) and pro-uPA (light gray bars) changes in HUVECs determined by fluorometric cell ELISA. Single bars represent mean values ± SD of at least 5 independent experiments. A statistically significant loss of the reactivity of the cells with antibodies specific for pro-uPA was seen after one hour of VEGF₁₆₅ or VEGF-E stimulation (30.2 ± 16.9% or 22.1 ± 20.4% of controls, both P < .01). The decrease to 77 ± 16.3% of initial total uPA reactivity was comparable with the decrease of uPAR reactivity (70.3 ± 5.2%). ^*P < .05; ^**P < .01 (n = 5). (C) Representative immunocytofluorimetric histograms of cell surface (gray) and total (black) uPAR in HUVECs after stimulation under serum-free conditions with 50 ng/mL VEGF₁₆₅ for 60 minutes or untreated as a control. VEGF₁₆₅ induced a decrease in cell surface uPAR of 29.7 ± 5.2%, whereby total uPAR levels were not affected, indicating an internalization process. (D) Effect of VEGF₁₆₅ (50 ng/mL) on pro-uPA activation on the surface of HUVECs measured by fluorimetric cell ELISA. Receptor bound urokinase antigen was removed by acid treatment as described by Stoppelli et al,³  following saturation of the free uPAR with exogenously added pro-uPA (25 mg/mL). Washed cells were stimulated with VEGF₁₆₅ (50 ng/mL) for 2 hours. Growth factor stimulation significantly decreases cell surface pro-uPA levels. P = .002.