Figure 6

Chrysin and its derivatives influence the calcium mobilisation and phosphorylation of various signalling proteins in platelets. Fluo4 AM-dye labelled human washed platelets (Ai,Aii) and platelet-rich plasma (Bi,Bii) were treated with vehicle control (V) [0.1% (v/v) DMSO] or different concentrations of chrysin (C) or Ru-thio-chrysin (Ru-tc) for 5 minutes prior to the addition of 0.5 μg/mL CRP-XL and monitoring of calcium mobilisation for three minutes by flourimetry. The calcium traces (Ai and Bi) shown are representative of three separate experiments. The maximum fluorescence obtained with each sample was converted into percentages to calculate the level of calcium mobilisation obtained with vehicle and chrysin or Ru-thio-chrysin treated samples. Cumulative data (Aii and Bii) represent mean ± S.D. (n = 3). *Indicates significance with respect to controls and ^#indicates significance with respect to the respective chrysin concentrations; p values shown (**^,## p < 0.01 and ***p < 0.001) are as calculated by one-way ANOVA using Graphpad Prism. (C) Human washed platelets were treated with vehicle control or different concentrations of chrysin or Ru-thio-chrysin for 5 minutes prior to the addition of 0.5 μg/mL CRP-XL and incubation for another 5 minutes. The platelets were lysed and used for immunoblot analysis to detect the level of AKT phosphorylation at residue S473, FAK phosphorylation at Y397 and phosphorylation of Src at Y527. The level of 14-3-3ζ protein was detected as a loading control. The cropped images of the blots shown here are representative of three separate experiments. The uncropped full length blots are presented in Supplementary Information (Figure S8).