Thrombin-induced DAF expression protects ECs against complement-mediated injury.
HUVECs were plated at confluence (5 × 105 cells/well) in 6-well dishes and cultured overnight at 37°C prior to stimulation with 10 U/mL thrombin or plain medium alone (unstimulated) for 24 hours. Following harvesting, ECs were incubated with the antiendoglin MoAb RMAC8 or plain medium alone for 30 minutes at 4°C. The ECs were then washed in HBSS/1% BSA prior to addition of up to 5% NHS for 3 hours at 37°C. Binding of C3 was detected by flow cytometry using FITC-conjugated rabbit antihuman C3. (A) Percent C3 binding ± SD (n = 3) to unstimulated (black bars) and thrombin-stimulated HUVECs (hatched bars), with binding to unstimulated ECs (RFI ± SD = 8.1 ± 2.7) shown as 100%. The negative control represents C3 binding in the presence of HIHS but without preincubation with MoAb RMAC8. (B) C3 binding (RFI ± SD, n = 3) on unstimulated (black bars) and thrombin-stimulated HUVECs (hatched bars) in the presence of MoAbs 1H4 (anti-DAF) and EN4 (anti-CD31), both at 50 μg/mL. (C) HUVECs plated at confluence (1.5 × 105 cells/well) in 24-well plates were cultured overnight prior to stimulation with 10 U/mL thrombin or plain medium alone for 24 hours. Following loading with calcein acetoxymethyl ester, ECs were incubated with MoAb RMAC8 for 30 minutes at 37°C. The ECs were then washed in HBSS/1% BSA prior to the addition of baby rabbit complement or HIHS for 45 minutes at 37°C. Calcein release was measured and percent cell lysis calculated as outlined in “Materials and methods.” The data are presented as percent cell lysis ± SD, n = 3. The figure is representative of 3 similar experiments performed on different EC cultures. * P < .05, ** P < .001.
