Differential modulation of Bruton's tyrosine kinase inhibitor selectivity on platelet <scp>GPVI</scp> and integrin <scp>αIIbβ3</scp> bidirectional signalling via <scp>BTK</scp>/<scp>PLCγ2</scp>/<scp>PKCθ</scp> affects haemostasis and thrombosis

The selectivity of Bruton's tyrosine kinase inhibitors (BTKi) affects the activation of platelet integrin αIIbβ3 differently, and this is related to the glycoprotein VI (GPVI) pathway. However, the distinct regulatory mechanisms remain to be further unravelled. In this research, we explored the impact of two BTKi with different selectivity, ibrutinib and zanubrutinib, on haemostasis and thrombosis by differentially modulating the bidirectional signalling of platelet integrin αIIbβ3. We found that both BTKi differentially regulate collagen-induced GPVI signalling, with ibrutinib more strongly inhibiting the activation of platelets and integrin αIIbβ3 than zanubrutinib. Clot retraction and laser confocal microscopy assays demonstrated that both BTKi can modulate clot retraction and platelet spreading via influence on integrin αIIbβ3 outside-in signalling, with ibrutinib showing a stronger inhibitory effect. Experiments involving tail bleeding and ferric chloride-induced thrombosis in rats reflected the differential impact on haemostasis and thrombosis. Notably, the downstream signalling of the integrin αIIbβ3 outside-in pathway is significantly influenced by ibrutinib, unlike zanubrutinib, through the BTK/PLCγ2/PKCθ pathway. These findings demonstrate that the selectivity of ibrutinib and zanubrutinib leads to different inhibitory effects on platelets and integrin αIIbβ3 activation. Enhanced inhibition of the BTK/PLCγ2/PKCθ signalling pathway by ibrutinib can increase the bleeding risk related to BTKi.