Defining the Effect of BTK Inhibitors on Haemostasis and Thrombosis

Abstract

ABSTRACT Bruton’s tyrosine kinase (BTK) is a cytoplasmic kinase that is a member of the Tec kinase family that has an essential role in B-cells and haematopoietic cells including platelets. In addition, BTK plays a role in ITAM-mediated platelet activation pathways. In terms of primary haemostasis, BTK is important in platelet activation pathways that lead to formation of a platelet plug. In contrast, secondary haemostasis involves the activation of clotting factors that also contribute to the formation of a blood clot. During this process, the damaged blood vessel releases thrombogenic agents. One of these components is thrombin, that activates clotting factors including fibrinogen to convert to fibrin to form a blood clot. BTK is involved in the GPVI/FcR gamma chain, CLEC-2 and FcgammaRIIa mediated signalling pathways that are activated when platelets are stimulated, and it is thought to play a role in the formation of this platelet plug. However, BTK plays an important role in activating normal B lymphocytes leading to maturation and proliferation. In B-cell lymphoproliferative disease such as chronic lymphocytic leukaemia (CLL), waldenstroms macroglobulinaemia (WM), mantle cell lymphoma (MCL), small lymphocytic lymphoma (SLL) and autoimmune disorders. BTK protein and mRNA are highly overexpressed compared to normal B cells leading to promotion of maturation, proliferation and apoptosis. As a result, covalent irreversible BTK inhibitors that target Cysteine 481 in kinase domain such as Ibrutinib and Zanubrutinib, have been developed to target and inhibit BTK to treat these conditions. Ibrutinib, a Bruton’s tyrosine kinase inhibitor, is successful in providing advances in B-Cell-associated CLL and related disease. However, concerns are raised against the BTK inhibitor-related bleeding events, which have led to cautious monitoring of patients on Ibrutinib. The pathophysiological aspects have suggested a decreased signalling of 1 GPVI/FcR gamma chain and CLEC-2 pathways and some kinases like HER2, ITK, JAK3, EGFR, and Tec. To investigate the potential causes of bleeding associated with BTK inhibitors, this study will examine the impact of Ibrutinib, the more selective BTK inhibitor Zanubrutinib, and the novel BTK degrader NX-2127. Unlike traditional BTK inhibitors, NX-2127 functions through targeted protein degradation, leading to the proteasomal degradation of BTK rather than its inhibition. This study aims to define the mechanisms of platelet glycoprotein receptor shedding, the effects of BTK inhibitors and degraders on primary and secondary haemostasis, and their roles in thrombus formation and growth. We hypothesise that BTK inhibitors, including Ibrutinib (BGB-1672) and Zanubrutinib (BGB-3111), as well as the BTK degrader NX-2127, have differential effects on haemostasis and thrombosis, with NX-2127 potentially displaying distinct effects due to its mechanism of action. The findings from this thesis demonstrated that Ibrutinib, but not Zanubrutinib or NX- 2127, induced significant platelet receptor shedding (GPIbα, GPV, GPIX, and αIIbβ3), contributing to reduced platelet adhesion and impaired thrombus formation. Functional assays showed that Ibrutinib, but not Zanubrutinib, altered clot firmness and platelet-dependent clot stability, while secondary haemostasis remained unaffected across all treatments. NX-2127 did not impair platelet function despite inducing BTK degradation. These results support the hypothesis that BTK inhibitors have differential effects on haemostasis and thrombosis, and suggest that BTK degrader NX-2127 may offer therapeutic advantages with a reduced bleeding risk.