Effect of ibrutinib on platelet responses to collagen, CRP, and VWF in vitro. (A) Washed platelets from healthy donors were treated or not with ibrutinib at the indicated concentration for 10 minutes and stimulated with different agonists (collagen, 3.3 µg/mL; CRP, 9 µg/mL; U46619, 5 µM; thrombin receptor-activating peptide, 50 µM; thrombin, 0.5 UI/mL). Platelet aggregation was assessed by turbidimetry and results, expressed as percentage of aggregation, are means ± standard error of the mean (SEM; collagen: n = 12; CRP and other agonists: n = 6). In parallel to aggregation, the effect of ibrutinib on platelet signaling in response to 1 minute stimulation with (B-C) collagen or (D) CRP was assessed by western blotting (whole platelet tyrosine phosphorylation pattern, PLCγ2 phosphorylation on Tyr-753, and Src phosphorylation on Tyr-416) or by flow cytometry for Btk phosphorylation on Tyr-223. (Inset) Fluorescence intensity (MFI) in (a) resting, (b) CRP-stimulated, and (c) CRP-stimulated ibrutinib-treated platelets. Western blots shown are representative of 3 independent experiments. Results of western blot quantification are means ± SEM of 3 to 6 independent experiments. (E) Effect of increasing doses of ibrutinib on collagen-induced platelet aggregation in PRP (n = 4, mean ± SEM). (F) Effect of ibrutinib on platelet adhesion on VWF under arterial flow conditions (4000 s⁻¹ or 180 dyn/cm²) in whole blood. Under these flow conditions, platelet adhesion was dependent on GPIb as verified by its complete inhibition by a monoclonal GPIb antibody (data not shown). Blood from healthy donors was preincubated for 30 minutes with 0.5 µM ibrutinib or dimethylsulfoxide. After 5 minutes of flow, firm platelet adhesion was quantified, after washing with phosphate-buffered saline containing Ca²⁺/Mg²⁺ at 4000 s⁻¹ for 1 minute, by measuring the platelet surface coverage values (means ± SEM from 3 independent experiments). *P < .05 and **P < .01. Scale bar: 50 µm.