Acalabrutinib and ibrutinib therapy cause dysfunctional GPVI-mediated platelet aggregation. PRP from patients receiving ibrutinib (Ibr; n = 6), acalabrutinib (Acal; n = 7), healthy donors (n = 9), or Btk inhibitor naïve CLL patients (n = 5) was loaded into 96-well microtiter plates containing lyophilized platelet agonists. Plates were shaken for 5 minutes at 37°C and aggregation was measure by light transmission to give an end point measurement. (A) Scatter plot of EC₅₀ values calculated from concentration response curves for ADP, U46619, epinephrine, TRAP-6, CRP-XL, and collagen. Each point represents the response of a patient receiving acalabrutinib (red triangle) or ibrutinib (green square); healthy donor (gray circle) or Btk inhibitor naïve CLL patient (white diamond). NR, patients that did not respond to an agonist at the highest concentration tested. Concentration response curves for GPVI agonists (B) CRP-XL and (C) collagen; each point represents the mean % aggregation values ± standard error of the mean (SEM). Significant differences relative to healthy donors were tested by 2-way ANOVA with the Turkey multiple comparisons test. Aggregation of washed platelets from healthy donors pre-treated with a range of (D) acalabrutinib or (E) ibrutinib concentrations and then stimulated with 3 to 0.01 µg/mL CRP-XL.