Direct Binding of Lyn to GPIbβ Transmits Two-Way GPIb-IX Signaling to Stimulate Platelet Activation and VWF Binding

BACKGROUND: In high shear rate blood flow, thrombosis often starts with platelet adhesion via the interaction between VWF (von Willebrand factor) and its platelet receptor, the GPIb-IX (glycoprotein Ib-IX) complex. GPIb-IX also induces intracellular signals, mediating VWF-induced platelet activation and enhancing thrombin- and collagen-induced platelet activation. GPIb-IX signaling requires SFK (Src family kinase) Lyn. However, it remains unclear how Lyn mediates GPIb-IX signaling, whether Lyn directly binds to GPIb-IX, and if it is possible to target this signaling node for developing novel antithrombotics. METHODS AND RESULTS: Lyn directly binds to GPIb (glycoprotein Ib) β subunit between amino acids 144 to 161 at the transmembrane/cytoplasmic domain interface, and this binding is critical for not only the extracellular GPIb-IX ligand–induced intracellular signaling but also transmits outbound signals enhancing VWF-GPIb-IX interaction. A small peptide, mPLβ, derived from the Lyn binding site in GPIbβ, inhibited VWF-induced or α-thrombin–induced GPIbβ-Lyn interaction, Lyn/SFK activation, stable platelet adhesion and aggregation, and, notably, reduced GPIb-mediated platelet adhesion to VWF even in the presence of integrin inhibitors. Biomembrane force probe study further shows that mPLβ reduces the VWF-A1 domain-GPIb adhesion frequency and inhibits force-induced A1-mediated intraplatelet calcium elevation. Importantly, mPLβ formulated as a high-loading peptide nanoparticle inhibited platelet adhesion/aggregation induced by collagen and in vivo arterial thrombosis with a mild effect in prolonging bleeding time. CONCLUSIONS: Direct binding of Lyn to GPIbβ mediates 2-way GPIb-IX signaling to activate platelets and modulate VWF-GPIb interaction. Lyn-mediated GPIb-IX signaling is critical for platelet adhesion and aggregation induced by VWF and collagen and in arterial thrombosis. Targeting the Lyn-GPIbβ interaction, thus, has the potential for treating GPIb-IX-dependent thrombosis.