Ibrutinib but not zanubrutinib treatment leads to loss of GPIb-IX-V complex and integrin α_IIbβ₃ from the platelet surface and reduced ex vivo thrombus formation on type I collagen during arterial flow in B-CLL. Washed B-CLL platelets (100 × 10⁹/L) on either untreated or ibrutinib or zanubrutinib or normal healthy donor platelets were labeled with anti-human CD42a, anti-human CD41a, or anti-human GPVI PE-conjugated antibodies. Flow cytometric analysis was used to determine GPIb-IX (A), integrin α_IIbβ₃ (B), and GPVI (C) expression. Results are expressed as the mean ± SEM from a single collection tested in replicate and represent 3 independent experiments, by Student t test. *P ≤ .05, **P ≤ .01, ***P ≤ .005. (D) Representative images of thrombus formation over type I collagen in arterial flow conditions for patients with B-CLL whole blood, either untreated or treated with ibrutinib or zanubrutinib vs healthy control over 6 minutes. Scale bar represents 20 μm. (E) Thrombus volume (in cubic micrometers) over time was calculated by thrombus area (in square micrometers) × thrombus height (in micrometers) for B-CLL patients, untreated or treated with ibrutinib, zanubrutinib, or healthy control. Results are expressed as the mean ± SEM from a single collection tested in replicate. (F) Washed human platelets (200 µL of 100 × 10⁹/L) were pretreated with 20 µM fluorogenic TACE substrate at 37°C for 20 minutes. Platelets were then incubated with either vehicle control, 20 µg/mL type I collagen, 0.5 µM ibrutinib, or 0.5 µM zanubrutinib at 37°C for 40 minutes. The cleavage of the TACE substrate was monitored at 320/405 nm using a Clariostar microplate reader. Results represent 3 independent experiments and are plotted as the mean ± SEM, by unpaired Student t test. **P ≤ .01, ***P ≤ .005.