Pneumolysin induces platelet death. (A) Kinetics of platelet viability. PBS was used as viability control and Triton X-100 to induce platelet death. Pneumolysin in increasing concentrations induced platelet death measured by reduced substrate turnover. (B) Platelet viability was maintained in the presence of polyvalent human immunoglobulin (human Ig [Privigen]; 1 mg/mL), polyclonal rabbit anti-pneumolysin (rabbit pAb; 10 µg/mL), or a monoclonal mouse anti-pneumolysin antibody (mouse mAb; 7.5 µg/mL) despite a high concentration of pneumolysin (300 ng/mL). (C) Thrombus formation on collagen in a flow chamber in the absence of pneumolysin was monitored by image acquisition at an interval of 10 seconds by fluorescence microscopy at a shear stress of 1000 s⁻¹. In the presence of pneumolysin, thrombus formation was impaired. (D) Thrombus formation in the presence of pneumolysin was restored by polyvalent immunoglobulin. Human Ig (Privigen) alone had no effect on thrombus formation (supplemental Figure 10). (E) Quantification of the percentage of surface area covered over time by thrombi in the presence of pneumolysin in different concentrations or nonactive pneumolysin mutants. Different concentrations of pneumolysin (Ply) are color coded: 300 ng/mL (red); 30 ng/mL (orange); 3.0 ng/mL (green). Pneumolysin^C428G without cytolytic activity (brown); pneumolysin^W433F with ∼10% cytolytic activity (blue). (F) Quantification of the effect of polyvalent immunoglobulin (human Ig (Privigen); 1 mg/mL) on restoring thrombus formation in the presence of pneumolysin (300 ng/mL). RLU, relative luminescence unit.