DGKε deficiency leads to a prothrombotic phenotype of ECs. (A) HUVECs were transfected with 20 nM of control siRNA or with DGKE siRNA at 10 nM or 20 nM, and tissue factor (TF) cell-surface expression was analyzed 48 hours later by flow cytometry. HUVECs stimulated for 4 hours with 100 U/mL TNFα were used as a positive control. A representative histogram is shown, and the bar graph illustrates the mean fold change in MFI (± SEM) from 4 independent experiments. **P < .01. (B) The concentration of TF was measured by ELISA in culture supernatants from HUVECs and HMECs transfected with control or DGKE siRNAs 48 and 72 hours after transfection. The bar graphs represent the mean concentration of TF (± SEM) from 4 independent experiments. (C) HUVECs were transfected with control or DGKE siRNAs and cultured for 48 hours. TNFα was added for the last 4 hours of cell culture, and the expression of TF at the surface of cells was then analyzed by flow cytometry. The bar graph represents the mean fold change in MFI (± SEM) vs control siRNA–transfected and unstimulated cells, from 3 independent experiments. **P < .01 and ***P < .001 vs control siRNA–transfected unstimulated cells, and ^#P < .05 vs control siRNA–transfected and TNFα-stimulated cells. (D) Adhesion of freshly isolated calcein-AM–stained human platelets to HUVECs transfected with control or DGKE siRNAs (20 nM) was assessed by immunofluorescence microscopy. Representative photomicrographs show adhering platelets (green) on HUVECs (magnification ×40). The bar graph represents the mean fold change in the number of adherent platelets (± SEM) from 4 independent experiments.