Effects of novel Btk and Syk inhibitors on platelet functions alone and in combination in vitro and in vivo

Background Inhibitors of tyrosine kinases downstream of the B-cell receptor, such as Bruton's tyrosine kinase (Btk) or Spleen tyrosine kinase (Syk), used alone or in combination are new therapeutic options in the treatment of B-cell malignancies. A challenge in the development of second-generation Btk inhibitors is to limit their side effects such as the increased bleeding risk. Considering the pivotal role of Syk in immunoreceptor tyrosine-based activation motif mediated platelet signaling, the impact of inhibiting this kinase on platelet functions is also worth analyzing. Objectives We investigated the effect of a novel Btk inhibitor, tirabrutinib, and a Syk inhibitor, entospletinib, alone and in combination on platelet signaling and functions in vitro and ex vivo. Methods Platelet aggregation, secretion, and signaling responses as well as thrombus growth under flow were analyzed in the presence of the inhibitors alone or in combination in vitro, at clinically relevant doses, and ex vivo in patients treated with these inhibitors in the context of a phase I trial. Results Although tirabrutinib alone had modest effects on platelet activation in vitro and ex vivo, entospletinib alone efficiently inhibited washed platelet aggregation in response to collagen. However, entospletinib weakly affected platelet activation in platelet-rich plasma, in whole blood and ex vivo. Importantly, the combination of tirabrutinib and entospletinib induced a significant decrease in platelet response to collagen in vitro and ex vivo correlating with mild bleedings reported in some of the treated patients. Conclusion These new results should contribute to improve the safety of these targeted therapies.