PGN–anti-PGN immune complexes activate the classical complement pathway to induce C5b-9 complex formation. (A) Normal serum, Ig-depleted serum, commercial serum that was C1q-, C8-, or Factor d–depleted, or serum to which 10 µg/mL neutralizing antibodies to C5 or MBL were added, was incubated with 10 μg/mL PGN at 37°C for 1 hour, and the reaction was stopped by 10 mM EDTA. The serum was added to wells of an ELISA plate precoated with aE11 antibody to capture activated C5b-9. After washing, C5b-9 was detected using biotin–anti-human C6 (clone 9C4) and streptavidin-HRP. (B) Plasma (100 μL) was depleted of anti-PGN antibodies by incubation with PGN (10 μg) overnight at 4°C and the PGN–anti-PGN immune complexes were removed by centrifugation. The supernatant was reconstituted or not with normal human IgG (500 µg/mL), and C5b-9 formation was determined by ELISA as described in panel A. *P < .05.; NS, not significant. (C) Washed platelets were stimulated with PGN preincubated with plasma (PGN + plasma) or PGN-treated plasma supernatant (PGN-treated plasma) at 37°C for 20 minutes. The cells were fixed, stained with Cy5 anti-human CD41 and FITC anti-human C5b-9 and viewed with a Zeiss LSM 510 confocal microscope at ×100 magnification. Arrows point to colocalized CD41⁺ platelets and FITC-C9. The results are representative of 3 similar experiments. *P < .05.