Inhibition of Btk Does Not Block Platelet Activation By GPVI: Studies with Ibrunitib, Acalabrutinib and Patients with X-Linked Agammaglobulinaemia

Background : Ibrutinib and acalabrutinib, inhibitors of the protein tyrosine kinase Btk, are used to treat B cell malignancies. They bind irreversibly to Cys481 of Btk preventing autophosphorylation and phosphorylation of other substrates including PLCγ2. Patients treated with ibrutinib have increased bleeding events which correlate with inhibition of platelet aggregation by the collagen and fibrin receptor GPVI. However, patients treated with acalabrutinib, and patients with mutations in their Btk gene (X-linked Agammaglobulinaemia: XLA), do not bleed excessively.

Aims: We sought to investigate this discrepancy between the two Btk inhibitors and patients with XLA.

Methods: Aggregometry, tyrosine phosphorylation and Ca²⁺ mobilisation were performed in washed platelets. Aggregometry was also performed with platelet rich plasma (PRP) from patients taking ibrutinib 420mg daily, acalabrutinib 100mg twice daily, a control treatment and XLA patients.

Results: We have compared the effect of the irreversible inhibitors of Btk, ibrutinib, acalabrutinib, and platelets from patients with XLA on activation by a high concentration (10 mg/ml) of the GPVI agonist collagen receptor peptide (CRP). Loss of activation of Btk, as verified by western blotting for autophosphorylation (Y223), and phosphorylation of PLCγ2 on tyrosines 759 and 1217, had no effect on aggregation to CRP. 10-20 fold higher concentrations of ibrutinib and acalabrutinib blocked aggregation to CRP. Inhibition was independent of Tec inhibition since (i) it was reversible on washout; (ii) there was no further reduction in PLCγ2 phosphorylation; (iii) concentrations of ibrutinib and acalabrutinib that blocked phosphorylation of Tec did not block aggregation.

Conclusions: Our results confirm that ibrutinib blocks GPVI-induced platelet aggregation in vitro and ex vivo but show that, contrary to current understanding, this is due to an unidentified off-target effect. We hypothesise that reducing the dose of ibrutinib given to patients would abolish its off-target effects and therefore reduce bleeding. This is supported by the fact that patients with XLA, and those taking acalabrutinib, do not experience abnormal bleeding.

Acknowledgment: this work was supported by the British Heart Foundation.