Effect of C3 inhibitors on hemolysis and C3 fragment deposition of PNH erythrocytes. (A) Dose-response curves from the in vitro hemolysis assay with Cp40 (red) and its long-acting derivatives PEG-Cp40 (blue) and Ac-Cp40-K-PEG (green). Lysis of PNH erythrocytes (y-axis) is expressed as a relative percentage of the lysis observed without any inhibitor (in each experiment, 100% represents the lysis observed in AcNHS) relative to the concentration of the C3 inhibitors (x-axis). Curves represent the mean of 10 experiments performed on samples obtained from 2 PNH patients; error bars represent standard deviations. (B) Flow cytometry assessment of C3 fragment deposition on the surface of erythrocytes from untreated PNH patients. Dot plots show intact erythrocytes as gated by physical parameters; CD59 (59-PE monoclonal Ab; y-axis) vs C3 fluorescein isothiocyanate (Ab14396 polyclonal Ab; x-axis). Scheme of erythrocyte populations and potential pattern of C3-fragment deposition on normal, type II, and type III PNH erythrocytes as observed in the fluorescence-activated cell sorter plots (top); unmanipulated fresh erythrocytes (negative control) and erythrocytes incubated in the presence of eculizumab (acidified ABO-matched serum from a patient on eculizumab, positive control for C3 deposition) (middle, from left to right); and erythrocyte pellets after incubation in acidified ABO-matched NHS in the absence of inhibitors (positive control for hemolysis) and in the presence of blocking concentrations of Cp40 and PEG-Cp40. Plots show representatives out of the 10 experiments (bottom, from left to right). (C) Schematic representation of activity spectrum of Cp40 in comparison with anti-C5 therapy. Eculizumab only blocks MAC formation and thus intravascular lysis, thereby enabling possible C3 opsonization of surviving erythrocytes and subsequent extravascular hemolysis. C3 inhibitors such as Cp40 prevent C3 activation upstream, thereby preventing both intravascular lysis and possible C3-mediated extravascular lysis via CR.