Loss of platelet function due to pneumolysin is prevented by immunoglobulins. (A) Prior to pneumolysin treatment intracellular Ca²⁺ of washed platelets was labeled with Fluo-4-AM for 30 minutes. After incubation with pneumolysin, the kinetics of Ca²⁺ release was measured and values are given as fold change compared with NaCl control. Different concentrations of pneumolysin (Ply) are color coded: 300 ng/mL (red); 30 ng/mL (orange); 3.0 ng/mL (light blue). Pneumolysin^C428G without lytic activity (brown) pneumolysin^W433F with ∼10% lytic activity (blue) did not cause Ca²⁺ release. (B) Platelet aggregation is typically directly proportional to an increase in light transmission. Only pneumolysin 300 ng/mL (red) and 30 ng/mL (orange) induced an increase in light transmission, but platelets where no longer responsive to 20 µM TRAP-6. Light transmission did not change by addition of buffer, pneumolysin 3 ng/mL, or the mutant pneumolysins, but platelets were still responsive to 20 µM TRAP-6. (C-D) Polyvalent human immunoglobulin (human IgG (Privigen); 1 mg/mL; green), polyclonal rabbit anti-pneumolysin (10 µg/mL; orange) and a monoclonal mouse anti-pneumolysin antibody (7.5 µg/mL; blue) prevented the effects of pneumolysin (300 ng/mL; red) in calcium influx (C) and platelet aggregation (D). In the presence of these immunoglobulins platelets became again responsive to 20 µM TRAP-6 (to enable comparison with the experiments without immunoglobulins, the data are shown here, although they are presented in the text at the end of “Results”).