Abstract
IntroductionBruton’s tyrosine kinase (BTK) plays a crucial role in the development and maturation of B-cells. A common side effect of Ibrutinib, a BTK inhibitor approved for the treatment of chronic lymphocytic leukaemia (CLL), is active bleeding in the absence of vascular injury. The mechanisms by which ibrutinib alters haemostasis however are currently unclear. The aim of this study was to investigate the effects of ibrutinib on platelet and endothelial cell function in vitro, to determine the mechanisms that underpin ibrutinib-induced bleeding.MethodsPlatelet rich plasma (PRP) collected from healthy volunteers was treated with increasing concentrations of ibrutinib for 15 min at 37°C, prior to stimulation with collagen (2 µg/ml) or ADP (10 µM). Platelet function and activation were measured by light transmission aggregometry (LTA) and flow cytometry respectively (CD62P+, PAC1+) and platelet morphology analysed using scanning electron microscopy (SEM). Platelet signalling pathways were analysed by Western blotting and the generation of endothelial microvesicles (EMVs) from HUVECs enumerated by flow cytometry, following 24 hours treatment with ibrutinib (increasing concentrations from 0.1 µM to 10 µM).ResultsIbrutinib significantly reduced collagen-mediated platelet aggregation and activation in a dose-dependent manner (p<0.05). SEM analysis also demonstrated that collagen-mediated shape change and filopodia formation was defective following ibrutinib treatment. Consistent with these findings, signalling downstream of the collagen GPVI receptor was perturbed, with a marked reduction in PLCγ2 phosphorylation. Ibrutinib only exerted mild inhibition of ADP-induced platelet aggregation (p<0.05), which was accompanied by reduced PLA2 activation and inhibition of VASP dephosphorylation. Additionally, our results demonstrated that at low concentrations, ibrutinib reduced the generation of pro-thrombotic EMVs, an effect that is reversed at the highest concentrations (p<0.05). ConclusionIbrutinib reduces collagen and ADP-mediated platelet aggregation, and activation by reducing phosphorylation of PLCγ2 and PLA2 and inhibiting VASP dephosphorylation. In addition, ibrutinib also appears to alter endothelial cell function by reducing EMV release. Understanding the mechanisms by which ibrutinib alters haemostasis may lead to the identification of novel antithrombotic targets.