CD62P expression of platelets by pneumolysin is caused by pore formation. Washed platelets of a defined set of 6 donors were incubated with various concentrations of pneumolysin (Ply). CD62P was detected by flow cytometry using antibodies against CD62P (P-selectin). The data are presented as GMFI of the positive gated events multiplied with the percentage of positive gated events in the dot plots. (A) PBS (gray) and phospholipase C (PLC; gray) from Staphylococcus aureus known to not activate platelets¹³  were used as negative controls and 20 µM TRAP-6 (gray) as positive control. (B) Pneumolysin (red; ng/mL) caused CD62P expression and dose-dependently inhibited an additional response to TRAP-6 (black). (C) Polyvalent human immunoglobulins (human IgG; Privigen) neutralized the effect of pneumolysin (pneumolysin plus human IgG = light blue) (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”). (D) Pneumolysin^C428G without lytic activity (brown) did not activate platelets or impaired the response to TRAP-6 and (E) pneumolysin^W433F with ∼10% lytic activity (purple) had a very minor effect only at 300 ng/mL. (F) Visualization of pore formation in the platelet membrane by pneumolysin by scanning electron microscopy. Platelets are altered in their shape and formed vesicles but not pseudopodias. At the left side, a platelet with pores can be seen. Inset, a higher magnification of the platelet indicating a pore by an arrow.