Strong CP activation leads to hemolysis despite of C3 peptide inhibitor Cp40. (A) CP- and AP-mediated lysis in presence of Cp40. A total of 80% NHS naturally containing antibodies against the Forssman antigen (which is present on shRBCs) was mixed with Cp40 to obtain final Cp40 concentrations of 0.25 to 16 µM, as indicated. AP activity was determined by mixing rabbit erythrocytes with 80% NHS supplemented with 5 mM Mg-EGTA in the presence of the same range of Cp40 concentrations. A serum mix with phosphate-buffered saline (PBS) instead of Cp40 served as positive control, whereas 5 mM EDTA and 5 mM Mg-EGTA dissolved in PBS (in case of CP; for each final concentration) served as negative controls (PBSE, PBS containing EDTA). Released hemoglobin was measured as a marker of hemolysis (average of 3 independent assays with standard deviation [SD] is shown). (B) ABO-incompatible human whole blood model. Human RBCs of blood group AB was added into human whole blood from 2 different donors with blood group O and incubated at 37°C for 3 hours in a reaction tube that had been surface treated with an anticoagulant. Absorbance of released hemoglobin was measured (after blank subtraction) at 415 nm as a marker of hemolysis. Final concentrations of analytes in the assay were: EDTA 10 mM (PBSE), eculizumab 1 µM (Ecu), Cp40 15 µM. The average of 6 independent assays with SD is shown. The observed absolute Abs (415 nm) ranges, after being corrected for the blank values, were for donor 1: EDTA, −0.01 to 0.17; Ecu, 0.03-0.55; Cp40, 0.06-0.60; for donor 2: EDTA, −0.03 to 0.07; Ecu, 0-0.19; Cp40: 0-0.17). An ordinary 1-way analysis of variance (ANOVA) using Tukey's multiple comparisons test was applied ns, not significant. (C) Forssman antigen induced activation of CP in presence of Cp40 at varying serum concentrations. Hemolysin-sensitized shRBCs were incubated in varying serum (containing antibodies against Forssman antigen) concentrations from 1.25% to 80%. Cp40 concentration in 80% serum was 16 µM and diluted accordingly, together with the serum resulting in a constant threefold molar excess of Cp40 over C3 in the serum. PBS, 5 mM EDTA, or Mg-EGTA served as controls as in panel A. Released hemoglobin was measured as a marker of hemolysis (average of 3 independent assays with SD is shown). (D) As in panel B, with the difference that shRBCs were sensitized with hemolysin before being exposed to NHS from which antibodies against the Forssman antigen had been preabsorbed. (E) C3/C4 surface opsonization after CP activation. shRBCs were assayed in 80% serum containing antibodies against the Forssman antigen in presence of C5 double inhibition (eculizumab and Coversin each at a final concentration of 0.5 µM to completely inhibit hemolysis). Then, cells were stained with an anti-human C3c mAb and C4c mAb. Fluorescence signals (median fluorescent index [MFI]) were measured by flow cytometry (average of 3 independent assays with SD is shown). (F) C3/C4 surface opsonization after AP activation. rRBCs were mixed with 80% preabsorbed NHS supplemented with 5 mM Mg-EGTA in presence of double C5 inhibition (by eculizumab and Coversin each at a final concentration of 0.5 µM) and the specified analytes. Cells were stained with an anti-human C3c mAb and C4c mAb. Detection and data presentation as in panel D.