Selective degradation of platelet BTK by PROTAC NX-5948 provides antithrombotic benefits without affecting hemostasis

ABSTRACT: Current antithrombotic therapies are effective in reducing thrombotic events but are limited by their associated risk of bleeding. Bruton tyrosine kinase (BTK) acts as a key signaling switch that drives platelet activation during thrombosis but is largely dispensable for routine hemostasis. It is an important nonredundant signaling mediator downstream of the glycoprotein VI and C-type lectin-like receptor 2 receptors and plays a key role in thrombosis with minimal involvement in hemostasis, making it an attractive antithrombotic target. Although BTK inhibitors effectively reduce thrombosis, their clinical use has been limited because of off-target effects. Protein degraders may overcome this limitation by enabling the ubiquitin proteasomal system to selectively target and degrade BTK. We here assessed the ability of the BTK degraders NX-2127 and NX-5948, currently in clinical trials for B-cell pathologies, to target platelet BTK for degradation. NX-2127 and NX-5948 induced concentration-dependent degradation of BTK in washed platelets, platelet-rich plasma, and whole blood. NX-5948 showed no hook effect and outperformed NX-2127 in potency, efficacy, and degradation kinetics. Proteomic analyses confirmed selective BTK degradation by NX-5948, with no evidence of major off-target effects. BTK degradation impaired collagen-related peptide (CRP)-mediated integrin αIIbβ3 activation, P-selectin expression, platelet aggregation, and in vitro thrombosis, with protease-activated receptor 1-mediated platelet function being left intact. Dosing mice with NX-5948 led to efficacious degradation of platelet BTK and impaired CRP-mediated, but not thrombin-mediated, ex vivo platelet function. In vivo, arterial thrombosis was markedly reduced, without an increase in bleeding time. Together, these results highlight NX-5948 as a potent, selective BTK degrader with antithrombotic potential and minimal hemostatic impact.