Inhibitors of the tyrosine kinase Btk have been proposed as novel antiplatelet agents. In this study we show that low concentrations of the Btk inhibitor ibrutinib block CLEC-2-mediated activation and tyrosine phosphorylation including Syk and PLCγ2 in human platelets. Activation is also blocked in patients with X-linked agammaglobulinemia (XLA) caused by a deficiency or absence of Btk. In contrast, the response to GPVI is delayed in the presence of low concentrations of ibrutinib or in patients with XLA, and tyrosine phosphorylation of Syk is preserved. A similar set of results is seen with the second-generation inhibitor, acalabrutinib. The differential effect of Btk inhibition in CLEC-2 relative to GPVI signalling is explained by the positive feedback role involving Btk itself, as well as ADP and thromboxane A2 mediated activation of P2Y12 and TP receptors, respectively. This feedback role is not seen in mouse platelets and, consistent with this, CLEC-2-mediated activation is blocked by high but not by low concentrations of ibrutinib. Nevertheless, thrombosis was absent in 8 out of 13 mice treated with ibrutinib. These results show that Btk inhibitors selectively block activation of human platelets by CLEC-2 relative to GPVI suggesting that they can be used at 'low dose' in patients to target CLEC-2 in thrombo-inflammatory disease.
Bibliographical noteFunding Information: This work was supported by British Heart Foundation (BHF) Programme grant (RG/13/18/30563), a BHF Clinical Fellowship to PLRN (FS/17/20/32738), a BHF Senior Basic Science Research Fellowship to AB (FS/19/30/34173), an AMS springboard grant to AYP (SBF002\1099) and the University of Birmingham’s Institute of Translation Medicine and Institute of Cardiovascular Sciences; SPW holds a BHF Chair (CH03/003). JAE is supported by the Deutsche Forschungsgemeinschaft (DFG grant: Eb177/13-1). Btk deficient DT40 cells, plasmid constructs and rabbit anti-Btk antibody were a kind gift from Dr Mike Tomlinson (University of Birmingham, UK). PLR Nicolson has received research grants from Rigel Pharmaceuticals, Novartis Pharmaceuticals and materials from Principia Biopharma.
- disorders of platelet function
- venous thrombosis
- molecular pharmacology
- Platelet Membrane Glycoproteins
- Platelet Activation
- Lectins, C-Type
- Blood Platelets
- Protein Kinase Inhibitors/pharmacology
ASJC Scopus subject areas