Contribution of tumor necrosis factor (TNF) receptor subtypes to prothrombotic effects in endothelial cells in vitro. (a) TNFα (5 ng/mL, 4 hours)-induced upregulation of P-selectin is significantly reduced after TNFR2 knockdown. Whereas TNFα-dependent prothrombotic changes in thrombomodulin and plasminogen activator inhibitor 1 (PAI-1) expression were only reduced after TNFR1 knockdown (b, c), single knockdown of TNFR1 and TNFR2 both significantly reduced TNFα-induced generation of tissue factor (d) and eventually acceleration of blood clotting induced by human microvascular endothelial cell (HMEC) lysates (i). (e-h) The contribution of secreted autocrine factors (as assessed by stimulation with the supernatant of TNFα (5 ng/mL, 4 hours)-treated HMECs) to upregulation of P-selectin, PAI-1, and tissue factor was not changed after specific TNF receptor knockdown, while reduction of thrombomodulin was inhibited after knockdown of TNFR2. (j) Knockdown of TNFR1 (left) and TNFR2 (right) was achieved by small interfering RNA (siRNA) and magnetofection and was confirmed on mRNA as well as protein level as measured by real-time polymerase chain reaction and Western blot after 24 and 48 hours, respectively. *Significantly different versus respective control at P <0.05. #Significantly different versus control-transfected cells treated with TNFα (n = 4 to 8 independent experiments). ctr., control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MFI, mean fluorescence intensity; Neg., negative; rel. to ctr., relative to control; resp. ctr., respective control; TNFR1, tumor necrosis factor alpha receptor subtype 1; TNFR2, tumor necrosis factor alpha receptor subtype 2.