The Effect of Btk Inhibitors on Haemostasis and Thrombosis
Authors: Dobie Gasim , Sze Daniel Man-yuen , Tam Constantine , Jackson Denise 
Published: October 2019

Abstract

Introduction
The Btk inhibitor, Ibrutinib (Imbruvica) which has proven to be efficacious in achieving remission of lymphocytosis and lymph node enlargement in B-CLL, it does have adverse side effects of bleeding, including major haemorrhages. The bleeding associated with Ibrutinib use is thought to be due to a combination of on-target Btk inhibition (as Btk is a key component of platelet GPVI signalling) as well as off targeted inhibition of other kinases including EGFR, ITK, JAK3 and Tec kinase. The major next generation Btk inhibitors in clinical development include Zanubrutinib (BGB-3111). Zanubrutinib shows improved selectivity for Btk compared with Ibrutinib, and thus may have reduced bleeding effects. Our study aims to determine in detail differential platelet effects between Ibrutinib and Zanubrutinib in human and mouse models using in vitro, exvivo and in vivo approaches.
Methods
Intravital microscopy was used to determine thrombus formation and growth after Btk inhibitors treatment in vitro and ex vivo using micro-slides or inside the mesenteric arterioles after injury by ferric chloride (FeCl3). Z-stack digital Axiocam mRm camera (Carl Zeiss) and Zeiss Axiovision software was used to capture images. Three dimensional (3D) deconvolved reconstructions of thrombi formed were analysed for surface coverage of platelet aggregates (μm2), thrombus height (μm) and thrombus volume (μm3). Flow cytometry analysis was also used to determine the release of agonist-induced platelet P-selectin exposure and dense granule after treatment with Btk inhibitors.
Results
In vitro experiments demonstrated that Btk inhibitors did not affect alpha or dense granule secretion mediated by GPCRs agonists, thrombin, PAR1 or PAR4. However, they inhibited alpha granule secretion mediated by GPVI selective agonists, CRP-XL or Rhodocytin. Ibrutinib inhibited human thrombus formation on type I collagen, fibrinogen or von Willebrand factor under arterial shear with 3 fold reduction whereas Zanubrutinib had no effect over a dose dependent range of concentrations. Ibrutinib treated PRP significantly delayed the kinetics of clot retraction at all-time points over the 2 hour time frame compared to Zanubrutinib treated and vehicle control. The studies also showed that Ibrutinib but not Zanubrutinib inhibited ex vivo human thrombus formation on type I collagen under arterial shear using B-CLL patient samples. The data demonstrated that treatment of C57BL/6 mouse whole blood with 0.5-2.0 µM of ibrutinib significantly inhibited thrombus growth on type I collagen under in vitro flow conditions whereas Zanubrutinib was comparable to the vehicle control. Consequently, 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.
Conclusion
Btk inhibitors used in the treatment of B-cell malignancies have differential effects on platelet function and thrombosis. Zanubrutinib is superior to ibrutinib as it showed no effect on platelet thrombus formation, thus reduces risk of bleeding.

Disclosures
Tam: AbbVie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Beigene: Honoraria.