PB0824 - BTKi of Varying Specificity are Potent Inhibitors of Platelet ITAM Signaling Pathways

Background: Bruton’s Tyrosine Kinase inhibitors (BTKi) are increasingly being recognized for their effect on platelets. Data have emerged indicating potential inhibition of the pathways related to the immunoreceptor tyrosine-based activation motif (ITAM) receptors GPVI, CLEC-2, and FcγRIIa, as well as a potential role for BTK downstream of GPIb signaling. Still uncertain, however, is whether the effects are due to an effect on BTK itself or related kinases.

Aims: To compare the impact of BTKis of various BTK specificity on various platelet activation pathways.

Methods: Blood from healthy volunteers was obtained with informed consent under an IRB-approved protocol. Washed platelets were prepared and treated with DMSO vehicle or one of five BTKi drugs (ibrutinib, zanubrutinib, acalabrutinib, remibrutinib, and rilzabrutinib) before activating platelets with collagen-related peptide (3 µg/ml CRP; GPVI pathway), fucoidan (100 µg/ml, CLEC-2 pathway), and ristocetin (0.5 mg/ml, GPIb pathway). To assess activation via FcγRIIa, we used a new monoclonal antibody (R6H8) that our lab recently produced. We used the PAR-1 activator peptide SFLLRN (T6, 25 µM) as a control. Aggregation was stopped with a perchloric acid buffer after 5 minutes and samples analyzed by SDS-PAGE and immunoblotting to quantify BTK protein and BTK phosphorylation.

Results: Aggregation induced by low-dose CRP was completely inhibited by the addition of all BTKi at 2 µM and all but acalabrutinib at 400 nM (Figure 1). Aggregation induced by fucoidan and by R6H8 were inhibited by all the BTKi at 400 nM, including acalabrutinib. Aggregation induced by T6 or ristocetin was not inhibited by the three BTKi tested. Immunoblot analysis displayed more prominent phosphorylation of BTK at position Y551 by CRP than by ristocetin (FIgure 2).

Conclusion(s): We conclude that BTKi of varying specificity inhibit platelet aggregation stimulated through ITAM pathways, supporting their potential for treating ITAM-mediated immunothrombosis, and thrombosis associated with heparin-induced thrombocytopenia and anti-phospholipid syndrome.