Figure 3.

Effect of ibrutinib and acalabrutinib on tyrosine phosphorylation events. (A) Washed platelets from healthy donors were treated or not with increasing doses of acalabrutinib (ACP) for 1 h at 37°C and stimulated with collagen 3.3 μg/mL. Platelet aggregation was assessed by turbidimetry during 10 min and results, expressed as percentage of maximal aggregation, are mean ± standard error of mean (SEM). Ten donors with low sensitivity (LS) and eight with high sensitivity (HS) to ibrutinib were analyzed. **P<0.01, according to one-way analysis of variance (ANOVA). Half maximal inhibitory concentrations (IC₅₀) were determined using GraphPad Prism software. (B) In parallel to aggregation, the effect of ibrutinib and acalabrutinib on platelet tyrosine phosphorylation events (PLCγ2 phosphorylation on Tyr-753 and Src phosphorylation on Tyr-418) in response to 1 min stimulation with collagen 3.3 μg/mL was assessed by western blotting. The results of the western blot quantification by densitometric analysis are shown as means ± SEM from ten independent experiments for LS and seven independent experiments for HS. *P<0.05, **P<0.01, according to one-way ANOVA. Representative western blots are shown for each group.