Influence of ponatinib on thrombosis propensity, vessel wall, and platelets. (A) Aged mice were untreated (UT) or treated with the indicated TKI at the concentration shown for 14 days by oral gavage twice daily. Data shown are the mean ± 95% CI for the time to carotid artery vessel occlusion for each of the treatment conditions. Each symbol in the graph represents the investigation of a single mouse. (B) The vessel wall and adventitia of ponatinib (Poni)-treated mice (3 or 15 mg/kg twice daily for 14 days) were examined for the presence of increased reactive oxygen species (anti-nitrotyrosine) and apoptosis (anti–caspase 3) by immunohistochemistry. The histology on the left represents mouse aorta cross sections examined on a Leica SCN 400 Slide Scanner equipped with a Hamamatsu line sensor color camera and a 40×/0.65 numerical aperture objective and reviewed by digital microscope (Leica Biosystems) at 10× magnification. The insets in the right lower part of each histologic panel are a fourfold increase in the size of the section of each tissue shown. The brown intracellular material seen is the presence of antibodies to nitrotyrosine or caspase 3, respectively, in the tissue of mice treated with Poni. Graphs represent the mean ± 95% CI of the percentage of stained cells to the total number of cells present per high-powered field, as determined by manual counting after treatment with 2 different concentrations of Poni shown. Each symbol on the graph represents analysis from 1 complete slide. (C-D) Washed platelets from UT (red bars) and Poni-treated (blue bars) (3 mg/kg per day) mice were examined for the threshold concentration for CRP-induced integrin activation (JON/A) and P-selectin expression (mean ± standard error of the mean [SEM] of 3 separate experiments with washed platelets from 2 mice per experiment. (E-F) Washed platelets from UT (red bars) and Poni-treated (blue bars) (3 mg/kg per day) mice were examined for the threshold concentration for human α-thrombin–induced integrin activation (JON/A) and P-selectin expression (mean ± SEM of 3 JON/A and 5 P-selectin experiments with washed platelets from 2 mice per experiment). (G) Aged mice were UT or treated with the indicated agent for 14 days by oral gavage. Poni treatment was at 3 mg/kg (twice daily) and pioglitazone (Pio) (10 mg/kg) was administered daily. Data shown are the mean ± 95% CI for the time to carotid artery vessel occlusion for each of the treatment conditions shown. Each symbol in the graph represents the investigation of a single mouse. P values were determined by Student t test. (H-I) Graphs represent the mean ± 95% CI of the percentage of stained cells for nitrotyrosine (H) or caspase 3 (I), respectively, to the total number of cells present after treatment with ponatinib (3 mg/kg twice daily by gavage) in the absence or presence (Poni and Pio) of simultaneous daily treatment with Pio (10 mg/kg). Preparation of slides and analysis were performed as described in panel B. Each symbol on the graph represents analysis from 1 complete slide. (J-K) Washed platelets from untreated (red bars), 3 mg/kg twice daily Poni-treated (blue bars), and combined Poni- and 10 mg/kg daily Pio-treated (light green bars) mice were examined for the threshold concentration for CRP-induced integrin activation (JON/A) and P-selectin expression (mean ± SEM of 5-6 separate experiments with washed platelets from 2 mice per experiment. *P < .0001, **P < .01, ***P < .025, ****P < .04. ns, not significant.