Heme activates the complement AP in serum. (A-C) Normal or FB-depleted sera was incubated v/v with twofold dilutions of heme in M199 medium, without or with 10 mM EGTA and 5 mM MgCl₂. After a 30-minute incubation at 37°C, the levels of released C3a, C5a, and sC5b9 were measured by ELISA. (D-F) To show the involvement of the AP, these complement components were measured after a similar incubation of NHS, without or with EGTA-Mg, C2-depleted (dpl) serum, or FB-depleted serum, reconstituted or not with 150 μg/mL purified FB, with 100 μM heme. Results are expressed as mean ± standard deviation (SD) of 2 experiments with triplicate measurements.