Investigating the impact of Bruton’s tyrosine kinase inhibitors used in the treatment of lymphoproliferative disease on haemostasis and thrombosis
Creators: name: DOBIE, Gasim
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 320102
subject: Haematology

description: The Bruton's tyrosine kinase (Btk) inhibitor, ibrutinib, has proven to be efficacious in the treatment of B cell chronic lymphocytic leukaemia (B-CLL) and related diseases.  However, a major adverse side effect of ibrutinib is bleeding including major haemorrhages.  The bleeding associated with ibrutinib use is thought to be due to a combination of on-target irreversible Btk inhibition as well as off targeted inhibition of other kinases including EGFR, ITK, JAK3 and Tec kinase. In this study, we investigated the effects of ibrutinib versus zanubrutinib (a more selective Btk inhibitor) on platelet function and thrombus formation on healthy human, B-CLL patients and on C57BL/6 mice using in vitro, ex vivo and in vivo approaches.  Both drugs (but particularly ibrutinib) reduced platelet aggregation responses to a range of agonists but did not affect alpha or dense granule secretion mediated by G-protein coupled receptor (GPCR) agonists, thrombin, PAR-1 or PAR-4. Both Btk inhibitors inhibited alpha granule secretion mediated by collagen receptor glycoprotein VI (GPVI) selective agonists, CRP-XL and Rhodocytin. Ibrutinib inhibited human thrombus formation on type I collagen, von Willebrand factor or fibrinogen under arterial shear stress conditions with a 3-fold reduction whereas zanubrutinib had no effect over a dose dependent range of concentrations. Ibrutinib treated platelet-rich plasma (PRP) significantly delayed the kinetics of clot retraction over time compared to zanubrutinib treated PRP and vehicle control. Treatment of whole blood from C57BL/6 with 0.5-2.0 µM of ibrutinib significantly inhibited thrombus growth on type I collagen under in vitro flow conditions whereas thrombus growth in the presence of zanubrutinib was comparable to the vehicle control. In addition, pre-treatment of C57BL/6 mice with ibrutinib (10 mg/kg), but not zanubrutinib (10 mg/kg) markedly inhibited platelet thrombus growth and formation on type I collagen under ex vivo arterial flow conditions. Intravital microscopy of vascular injury of mesenteric arterioles induced by ferric chloride (FeCl3) demonstrated that ibrutinib (10 mg/kg), but not zanubrutinib (10 mg/kg) inhibited in vivo murine thrombus formation and growth over time.  As determined by fluorescence activated cell sorting analysis of washed platelets, ibrutinib, but not zanubrutinib, induced a time and dose dependent shedding of GPIb-IX-V complex and integrin αIIbß3 but not GPVI from the platelet surface.  The shedding of GPIbα was blocked by a broad range metalloproteinase inhibitor and more importantly, by TAPI-2, an ADAM17 but not ADAM10 inhibitor.  Ibrutinib but not zanubrutinib treatment of human platelets increased ADAM17 activation.  Platelets from a proportion of ibrutinib-treated B-CLL patients showed reduced GPIb-IX-V complex and integrin αIIbß3 platelet surface expression which was not observed in zanubrutinib-treated patients.  Ibrutinib but not zanubrutinib treated mice led to increased soluble GPIbα (glycocalicin) and soluble αIIb levels in plasma.  These data demonstrate that ibrutinib induces shedding of GPIbα by an ADAM17-dependent mechanism and integrin αIIbß3 by an unknown sheddase, and that this process can occur in vivo to regulate thrombus formation.  Taken collectively, these data suggest that zanubrutinib has less effect on platelets and thrombus formation compared to ibrutinib which may translate to fewer haematological complications and therefore improved safety when used to treat B-CLL disease in pateints.