Acalabrutinib inhibits signal transduction downstream of GPVI with lower potency than ibrutinib. Washed human platelets from healthy donors were preincubated with a range of acalabrutinib or ibrutinib concentrations and then stimulated with 1 µg/mL CRP-XL for 3 minutes at 37°C and phosphorylation of (A) PLCγ2 (Y759) and (B) PKC substrates (S/T) was measured by western blotting. (C) Representative western blot images for PLCγ2 (Y759) and S/T phosphorylated PKC substrates. (D) Cytosolic Ca²⁺ following stimulation with 1 µg/mL CRP-XL was measured in fura-2 loaded platelets in real time for 5 minutes. Points represent the mean response relative to vehicle ± SEM. Representative [Ca²⁺]_i traces following incubation with (E) acalabrutinib or (F) ibrutinib.