Figure 4.

Chrysin and its derivatives inhibit inside-out and outside-in signalling in platelets. (A) human platelet-rich plasma was treated with vehicle [0.1% (v/v) DMSO] or diverse concentrations of chrysin, thio-chrysin, Ru-chrysin and Ru-thio-chrysin for 5 minutes prior to the addition of 0.5 μg/mL CRP-XL and incubation of 20 minutes at room temperature. The level of fibrinogen binding (as a marker for inside-out signalling to integrin αIIbβ3) was quantified using FITC-labelled anti-human fibrinogen antibodies by flow cytometry. The level of fluorescence obtained with vehicle control was taken as 100% to calculate the extent of inhibition in chrysin and its derivatives-treated samples. R represents ‘resting’ platelets. Cumulative data represent mean ± S.D. (n = 4). (B) human platelet-rich plasma was treated with vehicle (V) [0.1% (v/v) DMSO] or 50 μM of chrysin (C) or its derivatives, thio-chrysin (Tc), Ru-chrysin (Ru-c) and Ru-thio-chrysin (Ru-tc) for 5 minutes. Following incubation, clotting was initiated by the addition of 1 U/mL thrombin and the clot retraction was monitored for three hours. The remaining clot weight at three hours was measured to analyse the extent of retraction process. The image shown on the right is representative of four separate experiments. *Indicates significance with respect to controls and ^#indicates significance with respect to the respective chrysin concentrations; p values shown (*^,# p < 0.05, **^,## p < 0.01 and ***^,### p < 0.001) are as calculated by one-way ANOVA using Graphpad Prism.