FH binds to heme-treated C3 and is the major serum factor regulating C3 deposition on heme-treated cells. (A) Heme-exposed C3 bound more strongly to immobilized FH, as measured by SPR but (B) heme-exposed FH bound immobilized C3 in the same manner as did native FH. (C) HUVECs were treated with increasing doses of heme and 1:4 NHS, detached as in Figure 2A, and labeled with anti-FH mAb Ox23. Results are expressed as RFI mean ± SD (n = 3). (D) Resting HUVECs and cells treated with 100 μM heme for 20 minutes, were sequentially incubated for 30 minutes at 37°C, first with either PBS, FH-depleted serum, or C3-depleted serum (source of FH), washed, and incubated as a second step with NHS (*P < .05, paired Student t test; n = 3). (E) HUVECs treated with 100 μM heme for 20 minutes were incubated with increasing concentrations of Short Consensus Repeat (SCR) 19-20 FH peptide for 15 minutes at 37°C, then with 1:4 NHS for 30 minutes. (D-E) C3 deposits were analyzed by flow cytometry and expressed as mean ± SD RFI (n = 3).