Figure 2.
The effect of Btk inhibitors on thrombin-, PAR-1-, PAR-4-, CRP-XL- and rhodocytin-mediated α- and dense-granule exocytosis. Washed human platelets (100 × 109/L) were incubated with a range of doses (0.5-2.0 μm) of ibrutinib or zanubrutinib for 10 minutes at 37°C, and either unstimulated (resting) or stimulated by thrombin (0.25 U/mL) (A), PAR-1 (1.25 μM) (B), PAR-4 (25 μM) (C), rhodocytin (0.7 µg/mL) (D), or CRP (0.25 µg/mL) (E). The platelets were then labeled with the FITC-P-selectin CD62P monoclonal antibody. Flow cytometric analysis was used to determine platelet P-selectin exposure. Quinacrine-labeled platelets were untreated or treated with a range of doses (0.5-2.0 μM) of ibrutinib or zanubrutinib for 10 minutes at 37°C. Platelets then were either unstimulated (resting) or stimulated by selective agonist thrombin (0.25 U/mL) (F), PAR-1 (1.25 µM) (G), PAR-4 (50 µM) (H), rhodocytin 0.7 µg/mL (I), and CRP 0.25 µg/mL (J). Flow cytometry analysis was used to determine the release of agonist-induced dense granule. (K) PAC-1 binding was determined for vehicle, ibrutinib, or zanubrutinib treatment after stimulation of washed human platelets (100 × 109/L) with thrombin (0.25 U/mL), PAR-1 (1.25 µM), PAR-4 (25 µM), rhodocytin (0.7 µg/mL), and CRP (0.25 µg/mL). Statistical analysis was performed with the unpaired Student t test. Results are represented as mean fluorescence intensity (MFI) ± SEM from 3 independent experiments. *P ≤ .05, **P ≤ .01, ***P ≤ .001, ****P ≤ .0001.