Fig. 1
NETs directly induce aggregation of washed human platelets. a Representative traces showing dose-dependent aggregation of washed human platelets (WP, 3 × 108 /mL) in response to cell free-NETs. 0.1, 1 and 10% NETs refers to NETs constituting 0.1, 1 or 10% of final reaction volume, respectively (e.g. 10% NET is 1-volume NET solution to 9-volume platelets). Platelet aggregation was measured by light transmission aggregometer (Chrono-log). NETs were used at 10% of final reaction volume for following experiments*. b Progress time curve displaying % of NET-induced platelet aggregation which steadily increased over 20 min. c ATP/ADP release from NET-stimulated platelets. WP (1 × 108/mL) were incubated with NETs with a gentle shake at 37 °C for 1 and 10 min before adding Chrono-Lume reagent. Luminescence was measured using Enspire Multimode Plate Reader (PerkinElmer). NETs induced significant ATP/ADP secretion from platelets at 1 and 10 min. Results represent fold change in luminescence arbitrary absorbance unit relative to vehicle (PBS) control. Data expressed as mean ± SEM; **P < 0.01, ***P < 0.0001, N ≥ 5. d Platelet signalling in response to NETs was examined by Western blot analysis. WP (3 × 108/mL) were incubated with collagen (5 μg/mL), vehicle (PBS) or NETs for 3 min. Twenty μl of total cell lysate was analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and immunoblotted for phospho-Tyrosine (p-Tyr-1000). Equal loading was verified by α-actinin. Western blots indicate tyrosine-phosphorylated proteins in NET-stimulated platelets. *10% NET solution is prepared from 1 × 107/ml of PMA-activated neutrophils, however due to losses during preparation steps, 10% NET is estimated to contain histone/DNA complexes equivalent to 3.8 ± 1.2 × 106/mL neutrophils (Additional file 1: Figure S1). The elastase content present in a 10% NET reaction volume is 292 ± 172 pg/mL