Figure 1.
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) PneumolysinC428G without lytic activity (brown) did not activate platelets or impaired the response to TRAP-6 and (E) pneumolysinW433F 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.