RSP1141, a reversible non-covalent BTK inhibitor, suppresses GPVI-mediated platelet activation

Aims The glycoprotein VI (GPVI)–Bruton's tyrosine kinase (BTK) axis drives collagen-induced platelet activation and arterial thrombosis. Approved BTK inhibitors act irreversibly on Cys481, raising concerns over off-target kinase inhibition, C481S resistance, and bleeding. We aimed to identify a non-covalent, reversible BTK inhibitor with antiplatelet activity and to define quantitatively its selectivity within the platelet GPVI cascade. Materials and methods Fifty-five imidazo[4,5- b ]pyridine derivatives were screened using a BTK enzymatic assay and CRP-XL-induced aggregation of washed human platelets. The lead compound, RSP1141, was characterized by concentration–response immunoblotting of GPVI signaling nodes, Ca 2+ mobilization, functional assays, drug washout, 370-kinase profiling, C481S enzymatic assay, molecular docking, and a murine FeCl₃-induced arterial thrombosis and tail-bleeding model. Key findings RSP1141 inhibited BTK (IC₅₀ 27.9 nM) and CRP-XL-induced aggregation (IC₅₀ 173.6 nM). In intact platelets, RSP1141 inhibited BTK-pY223 (IC₅₀ 0.316 μM) and PLCγ2-pY753 (0.325 μM) at essentially identical concentrations, whereas SYK-pY525/526 (2.88 μM) and Src-family kinase-pY416 (3.96 μM) required 9.1- and 12.5-fold higher concentrations. Downstream PKC, Ca 2+ , granule secretion, integrin, and spreading responses were suppressed. RSP1141 retained activity against C481S BTK (IC₅₀ 141 nM), docking predicted no covalent Cys481 engagement, and platelet aggregation and BTK–PLCγ2 signaling recovered after washout. In mice, oral RSP1141 delayed FeCl₃-induced carotid occlusion at 10 mg/kg, whereas tail bleeding time was not detectably prolonged. Significance RSP1141 engages the BTK–PLCγ2 node with approximately 10-fold cellular selectivity over proximal GPVI kinases, inhibits reversibly, and is antithrombotic in vivo without detectable bleeding prolongation, providing a mechanistically differentiated antiplatelet lead.