Inhibition of Btk by Btk-specific concentrations of ibrutinib and acalabrutinib delays but does not block platelet aggregation mediated by glycoprotein VI

Ibrutinib and acalabrutinib are irreversible inhibitors of Bruton's tyrosine kinase (Btk) used in the treatment of B cell malignancies. They bind irreversibly to cysteine 481 of Btk, blocking autophosphorylation on tyrosine (Y) 223 and phosphorylation of downstream substrates including phospholipase-?2 (PLC?2). In the present study, we demonstrate that concentrations of ibrutinib and acalabrutinib that block Btk kinase activity as shown by loss of phosphorylation at Y223 and PLC?2 delay but do not block aggregation to a maximally-effective concentration of CRP or collagen. In contrast, 10- 20 fold higher concentrations of ibrutinib or acalabrutinib block platelet aggregation to GPVI agonists. Ex vivo studies on patients treated with ibrutinib, but not acalabrutinib, show a reduction of platelet aggregation to CRP indicating that the clinical dose of ibrutinib but not acalabrutinib is supramaximal for Btk blockade. Unexpectedly, low concentrations of ibrutinib inhibit aggregation to CRP in patients deficient in Btk. The increased bleeding seen with ibrutinib over acalabrutinib is due to off-target actions of ibrutinib that occur because of unfavourable pharmacodynamics.