Figure 2.

HNG inhibits platelet integrin αIIbβ3 activation, spreading and clot retraction. (A‐C) Human platelets were pre‐incubated with 10μM HNG, scramble‐HNG (10μM) or vehicle for 10 minutes at 37℃, and the fluorescence of FITC‐conjugated fibrinogen binding (A) or FITC‐conjugated PAC‐1 (B, C) was measured by flow cytometry after stimulated by 1μg/ml CRP or 0.05U thrombin. A representative histogram is shown. Statistical data were analysed using X geometric mean fluorescence or the percentage of gated cells. **P < .01, ****P < .0001, ordinary one‐way ANOVA, Dunnett's multiple comparisons test. (D) Effect of HNG on platelet spreading. 10µM HNG or scramble‐HNG‐treated platelets were placed on fibrinogen‐coated glass coverslips for 1 h at 37℃. After washing with PBS to remove non‐adherent platelets, adhered platelets were stained with TRITC‐labelled phalloidin. Images were obtained with an Olympus fluorescence microscope. Representative images are shown. Statistical data were calculated from the mean of the average surface area of individual platelets. *P < .05, **P < .01, N > 3, ordinary one‐way ANOVA, Dunnett's multiple comparisons test. (D) HNG impairs platelet clot retraction. After incubation with 10 µM HNG or vehicle, human platelets were stimulated with 20 µg/mL fibrinogen and 1 U thrombin and recorded at the indicated time‐point using a camera. The clot area was quantified by the ratio of clot area to platelet suspension area at different time‐points. *P < .05, unpaired t test