Figure 4. SLY-induced pore formation-dependent Ca²⁺ influx triggers PNC formation.

(A–C) rSLY induces Ca²⁺ influx in human platelets. The purified platelets marked with Fluo-8 were resuspended in HBSS (with 2 mM Ca²⁺) and rSLY/rSLY^P353V (1 μg/mL) or rSLY that was pretreated by cholesterol (10 μg/mL). The Ca²⁺ influx in platelets was observed using an FV1000 confocal laser scanning microscope. The Ca²⁺ influx in platelets was observed using an FV1000 confocal laser scanning microscope. The following mean fluorescence intensity (MFI) of Ca²⁺ mobilization was recorded by Series Analysis (XY-T) software in FV1000. C1-C10, cell 1-cell 10. The video of these results were displayed in Supplementary Information. (D) The EGTA effect on rSLY-induced CD62P release from platelets in human blood was assessed by flow cytometry. (E) The EGTA (3 mM) effect on S. suis supernatant-induced PNC formation was detected by flow cytometry. THB and PBS are the negative controls for culture supernatant and proteins, respectively. EGTA was dissolved in H₂O. Data in B and C are given as the mean ± SD of three independent experiments from three different blood donors. **P < 0.01; ns, no significance; Cho, cholesterol; rSLY, recombinant SLY; 05ZYH33, wild type strain; ∆sly, isogenic sly mutants; Sup, supernatant.