GPIbα binds to S100A8/A9and increases VWF-dependent platelet agglutination. (A) Binding of rGPIbα to immobilized S100A8/A9. Data presented as mean ± SEM (n = 3). (B-D) Washed human platelets (106/condition) were preincubated with or without AK2 Ab (40 μg/mL), SZ2 (40 μg/mL), or both for 10 minutes at 37°C and then stimulated with S100A8/A9 (20 μg/mL) for 30 minutes at 37°C. Platelet activation was determined by flow cytometry using (B) anti-CD41/CD61 PAC-1 antibody, (C) anti–P-selectin antibody, and (D) annexin-V binding. Data are shown as MFI of treated platelets over control (unstimulated) for different markers. (E-G) Ristocetin-induced platelet aggregation was assessed by addition of VWF (1 μg/mL) and ristocetin (1.5 mg/mL) to washed platelets. Platelets were primed with S100A8/A9 and ristocetin for 6 minutes before addition of VWF. Platelet aggregation was monitored for 6 minutes after VWF addition (n = 6). (E) Representative trace. Data are shown as mean ± SEM. The statistical significance was analyzed using ordinary one-way ANOVA (B-D) and nonparametric Mann–Whitney t test (Kruskal-Wallis test) (F-G). ∗P < .05, ∗∗P < .01, ∗∗∗P<.001, ∗∗∗∗P < .0001.
