Neutrophil extracellular traps participate in the development of cancer-associated thrombosis in patients with gastric cancer

Figure 6 NETs drive hypercoagulation of ECs. A: ECs were cocultured with NETs (μg DNA/mL) or PBS in the presence of DNase I, APC, or sivelestat alone or together for 4 h and analyzed by confocal microscopy. The intercellular junctions of ECs were stained with VE–cadherin and phalloidin. Magnification 63×; scale bars: 10 μm. Red-phalloidin, Green-VE, and Blue-DAPI; B: EC activation was stained with CD31 and TF. Magnification 63×; scale bars: 10 μm. Red-TF, Green-CD31, and Blue-DAPI; C and D: VE–cadherin expression and TF expression on ECs were detected by confocal microscopy and analyzed with ImageJ software (expression indicated as MFI). MFI was defined as the ratio of total fluorescence intensity to the area; E and F: EC monolayers were stimulated with various concentrations of NETs for 4 h, followed by determination of fibrin formation by turbidity measurement at 405 nm, and the TAT complex level was detected by ELISA. All values are the mean ± SD. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001; ^dP < 0.0001. NETs: Neutrophil extracellular traps; ECs: Endothelial cells; DNase I: Deoxyribonuclease I; APC: Activated protein C; TF: Tissue factor; TAT: Thrombin–antithrombin; MFI: Mean fluorescence intensity.