Figure 5.
The effect of ibrutinib and zanubrutinib on in vitro, ex vivo, and in vivo thrombus formation and tail bleeding time. (A-B) Fluorescently labeled whole blood of C57BL/6 mice was untreated or treated with 0.5 μM of ibrutinib or 0.5 µM of zanubrutinib and was perfused over 500 μg/mL type I collagen under arterial flow conditions at a shear rate of 1800 seconds−1. Z-stack images were captured over a 6-minute duration with a digital AxioCam MRm camera (Zeiss) and analyzed with Zeiss Axiovision Rel 4.6 software. 3D deconvolved reconstructions of thrombi formed were analyzed for surface coverage of platelet aggregates (in square micrometers), thrombus height (in micrometers), and thrombus volume (in cubic micrometers). (A) Representative images of thrombi formation over type I collagen under arterial flow conditions for wild-type mouse whole blood treated with 0.5 μM of ibrutinib or 0.5 µM of zanubrutinib vs DMSO control over 6 minutes. Scale bar represents 20 μm. (B) Thrombus volume over time was calculated from thrombus area × thrombus height. Results are cumulative data from 4 independent experiments and presented as the mean ± SEM, by the unpaired Student t test. ***P ≤ .005, ****P ≤ .001. (C-D) Fluorescently labeled whole blood of C57BL/6 mice untreated or treated with 10 mg/kg ibrutinib or 10 mg/kg zanubrutinib was perfused over 500 μg/mL type I collagen under arterial flow conditions at a shear rate of 1800 seconds−1. Z-stack images were captured over a 6-minute duration with a digital AxioCam MRm camera (Zeiss) and analyzed with Zeiss Axiovision Rel 4.6 software. 3D deconvolved reconstructions of thrombi formed were analyzed for surface coverage of platelet aggregates, thrombus height, and thrombus volume. Scale bar represents 20 μm. (E) Representative images of thrombus formation over type I collagen under arterial flow conditions for wild-type mice treated with 10 mg/kg ibrutinib or 10 mg/kg zanubrutinib vs wild-type treated with DMSO over 10 minutes. Scale bar represents 20 μm. (F) Thrombus volume over time was calculated from thrombus area × thrombus height for all mice treated with ibrutinib, zanubrutinib, or DMSO. (G) Tail bleeding times for wild-type or ibrutinib- or zanubrutinib-treated mice. (H) Volume of blood lost (in microliters) in wild-type or ibrutinib- or zanubrutinib-treated mice. (I) Percentage of unstable hemostasis determined by rebleeding occurring 1 minute after initial clot formation for wild-type or ibrutinib- or zanubrutinib-treated mice. Results are cumulative data from 4 independent experiments and are expressed as the mean ± SEM. **P ≤ .01, ***P ≤ .001, ****P ≤ .0001.

The effect of ibrutinib and zanubrutinib on in vitro, ex vivo, and in vivo thrombus formation and tail bleeding time. (A-B) Fluorescently labeled whole blood of C57BL/6 mice was untreated or treated with 0.5 μM of ibrutinib or 0.5 µM of zanubrutinib and was perfused over 500 μg/mL type I collagen under arterial flow conditions at a shear rate of 1800 seconds−1. Z-stack images were captured over a 6-minute duration with a digital AxioCam MRm camera (Zeiss) and analyzed with Zeiss Axiovision Rel 4.6 software. 3D deconvolved reconstructions of thrombi formed were analyzed for surface coverage of platelet aggregates (in square micrometers), thrombus height (in micrometers), and thrombus volume (in cubic micrometers). (A) Representative images of thrombi formation over type I collagen under arterial flow conditions for wild-type mouse whole blood treated with 0.5 μM of ibrutinib or 0.5 µM of zanubrutinib vs DMSO control over 6 minutes. Scale bar represents 20 μm. (B) Thrombus volume over time was calculated from thrombus area × thrombus height. Results are cumulative data from 4 independent experiments and presented as the mean ± SEM, by the unpaired Student t test. ***P ≤ .005, ****P ≤ .001. (C-D) Fluorescently labeled whole blood of C57BL/6 mice untreated or treated with 10 mg/kg ibrutinib or 10 mg/kg zanubrutinib was perfused over 500 μg/mL type I collagen under arterial flow conditions at a shear rate of 1800 seconds−1. Z-stack images were captured over a 6-minute duration with a digital AxioCam MRm camera (Zeiss) and analyzed with Zeiss Axiovision Rel 4.6 software. 3D deconvolved reconstructions of thrombi formed were analyzed for surface coverage of platelet aggregates, thrombus height, and thrombus volume. Scale bar represents 20 μm. (E) Representative images of thrombus formation over type I collagen under arterial flow conditions for wild-type mice treated with 10 mg/kg ibrutinib or 10 mg/kg zanubrutinib vs wild-type treated with DMSO over 10 minutes. Scale bar represents 20 μm. (F) Thrombus volume over time was calculated from thrombus area × thrombus height for all mice treated with ibrutinib, zanubrutinib, or DMSO. (G) Tail bleeding times for wild-type or ibrutinib- or zanubrutinib-treated mice. (H) Volume of blood lost (in microliters) in wild-type or ibrutinib- or zanubrutinib-treated mice. (I) Percentage of unstable hemostasis determined by rebleeding occurring 1 minute after initial clot formation for wild-type or ibrutinib- or zanubrutinib-treated mice. Results are cumulative data from 4 independent experiments and are expressed as the mean ± SEM. **P ≤ .01, ***P ≤ .001, ****P ≤ .0001.

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