Ibrutinib treatment leads to loss of GPIbα, GPIX, and integrin α_IIbβ₃, but not GPVI, GPV, or GPIbβ from the platelet surface in a time- and dose-dependent manner. (A-K) Washed human platelets (100 × 10⁹/L) were incubated to determine dose response (dose range, 0.1-1.0 µM) of ibrutinib or zanubrutinib or vehicle control for 5, 10, 30, and 60 minutes at room temperature (RT). The platelets were then labeled with anti-human CD42a (GPIX), anti-human CD42b (GPIbα), anti-human CD41a (integrin α_IIb), anti-human CD61 (integrin β₃), anti-human GPIbβ (CD42c), or anti-human GPVI PE-conjugated antibodies or anti-human GPV (CD42d) APC-conjugated antibodies. (L-N) Washed human platelets (100 × 10⁹/L) were incubated with 100 µM GM6001 or 100 µM TAPI-2 for 2 hours at 37°C, followed by 0.5 µM ibrutinib treatment for 60 minutes at RT. The platelets were then labeled with anti-human CD42b, anti-human CD42a, or anti-human CD41a PE-conjugated antibodies. (O-P) Washed human platelets (100 × 10⁹/L) were incubated with 100 µM GI254023, 100 µM calpeptin, 100 µM eptifibatide, or 100 µM Dynasore hydrate for 2 hours at 37°C followed by 0.5 µM ibrutinib treatment for 60 minutes at RT. The platelets were then labeled with anti-human CD42b or anti-human CD41a PE-conjugated antibodies. Flow cytometric analysis was used to determine the expression of GPIbα or integrin α_IIbβ₃. Statistical analysis was performed with the unpaired Student t test. Results are expressed as mean fluorescent intensity (MFI) ± SEM from 3 independent experiments. ****P ≤ .0001.