Fig. 5
Syk, but not NOX1, inhibition attenuated NET-induced platelet aggregation. a Representative aggregation traces showing the effect of Syk phosphorylation (BAY61, 5 μM) and NOX1 (ML171, 5 μM) inhibitors on NET-induced platelet aggregation. WP (3 × 108/mL) were pre-incubated with the inhibitors for 15 min at 37 °C before addition of NETs. Platelet aggregation was measured using transmission aggregometer (Chrono-log). b Bar graph comparing the effect of BAY61 and ML171 on NET-induced platelet aggregation. Platelet aggregation percentage was calculated after 20 min on a light transmission aggregometer. Results were normalized for each donor relative to NET-induced platelet aggregation. Data are expressed as mean ± SEM, ***P < .001; ns: non-significant, n = 7. (c-d) Bar graphs comparing the effect of BAY61 (5 μM) and ML171 (5 μM) on platelet activation (measured by P-selectin and active αIIbβ3 expression, n = 3) and (e-f) ATP/ADP secretion (n = 6) elicited by NETs. Results were normalized for each donor relative to NET-induced platelet response. Data are expressed as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ns: non-significant. e Syk phosphorylation is augmented by NETs. WP (3 × 108/mL) were incubated with collagen (5 μg/mL), vehicle (PBS) or NETs for 3 min. Forty-five μl of total cell lysate was then analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and immunoblotted for p-Syk, p-Akt and p-Erk1/2. Equal loading was verified by α-actinin. In all assays, NETs constituted 10% of final reaction volume and contains 292 ± 172 pg/mL of NET-elastase. The immunoblots are representative sample of 3 independent experiments