Bruton’s tyrosine kinase (BTK) plays an important role in platelet signalling downstream of GPVI receptor and has been proposed as a target to prevent thrombosis in high-risk patients. However, current, clinically used BTK inhibitors (e.g. ibrutinib), have off target effects and are associated with an increased risk of bleeding. There is therefore a need to develop novel antiplatelet agents. This thesis is focussed on the targeted degradation of BTK in human platelets by using recently developed small molecule degraders (PROteolysis TArgeting Chimeras, PROTACs) that utilise the proteasomal system to degrade BTK. Human platelet rich plasma (PRP) was treated with various concentrations of PROTACs and were subjected to immunoblotting and assessed for integrin αIIbβ₃ activation and P-selectin expression in response to the GPVI and PAR agonists CRP-XL or α-thrombin, respectively. The multityrosine-kinase degrader TL12-186 and the BTK degraders DD04-15 and DD03-171 show potent BTK degradation, which can be overcome by the addition of excess amount of pomalidomide and MLN4924 (an NAE-inhibitor) to interfere with the ubiquitination process. GPVI-mediated integrin αIIbβ₃ activation and P-selectin expression were significantly impaired, whereas PAR-mediated responses were unaffected. BTK plays an important role in the activation of phospholipase C 𝛾2 (PLC𝛾2) which in turn plays a regulatory role in Ca²⁺ homeostasis of platelets during activation. Findings of this thesis show decreased CRP-XL-induced intracellular Ca²⁺ mobilisation in BTK-specific PROTAC-treated platelets. Tandem Mass Tag (TMT) proteomic approach was used to provide evidence for the specificity of the PROTACs DD04-015 and DD03-171 in degrading BTK compared to other kinases. These data confirm the high susceptibility of human platelets to BTK degraders, showing that PROTACs can be successfully utilised in modulating and studying human platelet function.
Proteolysis Targeting Chimeras: proteasomal degradation of platelet BTK and TEC as a novel approach in thrombosis prevention
Munkacsi, A. (Author). 6 Dec 2022
Student thesis: Master's Thesis › Master of Science by Research (MScR)