Vicinal disulfide bond modulates A1-GPIbα interaction by altering the force-induced conformational changes of A2 domain

Von Willebrand Factor (VWF) plays a crucial role in hemostasis and thrombosis, with its A1 and A2 domains being keys to its function. This study investigates the impact of the vicinal disulfide bond in the A2 domain on the interaction between the A1 domain and platelet glycoprotein Ibα (GPIbα). Using single-molecule optical tweezers, we evaluated the mechanical unfolding and refolding behaviors of wild-type A2 (WT A2) and an A2 mutant without the vicinal disulfide bond (CC-AA A2). Our results reveal that CC-AA A2 exhibits heterogeneous unfolding patterns and altered refolding characteristics compared to WT A2. Furthermore, microscale thermophoresis and AlphaFold3 predictions demonstrate enhanced binding affinity of CC-AA A2 to the A1 domain and decreased binding affinity of CC-AA A1A2 to the ligand binding domain (LBD) of GPIbα. These findings advance our understanding of the regulatory role of the vicinal disulfide bond in VWF function and its potential implications in hemostasis and thrombosis.