Forceful AP activation reveals TP activity in C5-inhibited serum on PNH cells. (A) Protection of PNH cells from complement alternative pathway mediated lysis. PNH-RBCs were incubated for 24h in acidified human serum mixed with complement inhibitors (75% final serum concentration; average of 3 independent assays with SD). (B) As in panel A, but with addition of Mg-EGTA (whereby EGTA chelates Ca ions to block CP activity) to the serum, with an incubation time at 37°C for 2 hours. Hemolysis was measured by determining the release of hemoglobin at 405 nm (1 typical assay out of 3 independent assays with average values of triplicate data points with SD is shown; for the other 2 assays, see supplemental Figure 5 A-B). (C) Residual hemolysis of PNH-RBCs preloaded with C3b in C5-inhibited serum. PNH-RBCs were preloaded with C3b in human factor I–depleted serum under combined C5 inhibition (with eculizumab and coversin) to allow for efficient C3b opsonization without lysis. C3b preloaded cells were washed and then exposed to 75% human serum in absence or presence of the inhibitors. The C3b-preloaded PNH cells were incubated for 2 hours in acidified human serum supplemented with 5 mM Mg-EGTA (1 typical assay out of 3 independent assays with average values of duplicate data points with SD is shown; for the 2 other assays, see supplemental Figure 5C-D). Ecu, eculizumab; HIS, heat-inactivated serum.