The melanoma cell line Ret induces endothelial cell stimulation via VEGF-A. HUVECs were stimulated for 15 minutes with the supernatant (sn) of the melanoma cell line Ret alone or supplemented with 0.65 mg/mL bevacizumab and thrombin (0.5 IU/mL) was used as a positive control. The efficiency of the melanoma cell-induced EC stimulation was quantified by measurement of VWF release by immunofluorescence staining (A) and by ELISA for VWF (B). Supplementation of Ret cells with bevacizumab or tinzaparin (100 IU/mL) reduced tumor cell-induced VWF release (B). Measurement of VEGF-A revealed that incubation with tinzaparin for 24 hours, 48 hours, and 72 hours resulted in a significant reduction of VEGF-A release by melanoma cells (C). The VEGF-A mRNA expression measured by real-time PCR was not affected by tinzaparin treatment of 48 hours (D). Bio-plex assays of different cell fractions showed a tinzaparin-induced reduction of VEGF-A in all fractions (E). Adding tinzaparin to Ret cell supernatant immediately before the measurements revealed an interaction of VEGF-A and tinzaparin (F). Binding of tinzaparin to VEGF-A was determined using the intrinsic tryptophan fluorescence (emission: 290 nm, excitation: 340 nm). The fluorescence of VEGF-A increases in a dose-dependent manner (G). In contrast to fondaparinux, tinzaparin exhibits a high binding affinity to VEGF-A (H). The binding of VEGF-A to tinzaparin inhibited VEGF-mediated ATP production of endothelial cells indicative for reduced cell proliferation (I). Data are presented as the mean ± SD of n = 4 of at least 2 independent experiments (*P < .05, **P < .005, ***P < .001; scale bars = 20 µm).