The effect of ibrutinib and zanubrutinib on in vitro human thrombus formation and growth. Fluorescently labeled whole human blood was either untreated or treated with 0.5 µM ibrutinib or 0.5 µM zanubrutinib at 37°C for 30 minutes before perfusion over 500 µg/mL type I collagen, 50 µg/mL VWF, or 100 µg/mL fibrinogen, under arterial flow conditions at a shear rate of 1800 seconds⁻¹. Z-stack images were captured over a 6-minute period with a digital AxioCam MRm camera (Zeiss) and analyzed with Zeiss Axiovision Rel 4.6 software. 3D deconvolved reconstructions of the thrombi formed were analyzed for surface coverage of platelet aggregates (in square micrometers), thrombus height (in micrometers), and thrombus volume (in cubic micrometers). (A,C,E) Representative images of thrombus formation over type I collagen, VWF, or fibrinogen, in arterial flow conditions, in human whole blood treated with 0.5 µM ibrutinib, 0.5 µM zanubrutinib, or vehicle control over 6 minutes. Scale bars represent 20 μm. (B,D,F) Thrombus volume over time was calculated from thrombus area × thrombus height for each Btk inhibitor. Results are cumulative data from 4 independent experiments and are presented as the mean ± SEM by unpaired Student t test. **P ≤ .01, ***P ≤ .001, ****P ≤ .0001.