Figure 4.

Effect of flavonoids on thrombin-, PAR₁-AP- and PAR₄-AP-induced tyrosine phosphorylation. Washed platelets were incubated with vehicle (dimethylsulphoxide), flavonoids (100 µM), the Src kinase inhibitor PP2 (10 µM) or the multiple kinase inhibitor staurosporine (1 µM), and stimulated with 0.5 U·mL⁻¹ thrombin, 100 µM PAR₁-AP or 500 µM PAR₄-AP for 2 min. Platelets were then lysed, and phosphotyrosine-containing proteins were identified by Western blot using the 4G10 antibody. A representative blot is shown in the upper panels. In each case, tyrosine phosphorylation was quantified by densitometry using Quantity One software. The lower panel shows comparative results of tyrosine phosphorylation achieved with platelets stimulated in the absence or presence of flavonoids or inhibitors. Data are mean ± SEM from three separate experiments. NA, non-activated; API, apigenin; GEN, genistein; QUE, quercetin; RUT, rutin; STA, staurosporine. *P < 0.05 compared with phosphotyrosine content in stimulated platelets in the absence of inhibitors.