Figure 1.
HNG impairs platelet aggregation and granule release. (A) Gel‐filtered human platelets were pre‐incubated with HNG (4 μM, 8μM or 10 μM) or vehicle solution (ddH2O) for 10 minutes and stimulated with collagen 2 µg/mL, thrombin 0.01 U/mL, convulxin 0.2 nM, ADP 10 μM and CRP 0.2 µg/mL, respectively. Statistical data of HNG10μM and vehicle were shown. *P < .05, **P < .01, ***P < .001, ****P < .0001, N > 3, unpaired t test. (B, C) The surface expression of P‐selectin was analysed by flow cytometry. 10 μM HNG or scramble‐HNG was pre‐incubated with gel‐filtered human platelets for 10 minutes at 37℃ and then stimulated by 2 μg/mL CRP (B) or 0.05U thrombin (C). A representative fluorescent histogram of PE‐conjugated P‐selectin is shown. Statistical data were analysed using X geometric mean fluorescence and the percentage of gated cells. ***P < .001, ****P < .0001, ordinary one‐way ANOVA, Dunnett's multiple comparisons test. (D) ATP released from platelets was monitored using an aggregometer. After incubation with HNG or vehicle, gel‐filtered platelets were induced by 2 µg/mL collagen or 0.2 nM convulxin. ATP standard (final concentration: 2 nM) was used to normalize the released ATP. *P < .05, mean ± SEM, N > 3, paired t test