The membrane-proximal intermolecular disulfide bonds in glycoprotein Ib influence receptor binding to von Willebrand factor

Abstract

Background: In the platelet glycoprotein (GP)Ib-IX complex, the binding site for its ligand von Willebrand factor (VWF) is restricted to the N-terminal domain of the GPIbβ subunit. How the other subunits in the complex, GPIbβ and GPIX, regulate the GPIbβ-VWF interaction is not clear. Objectives and methods: As GPIbβ connects with two GPIbβ subunits via disulfide bonds, we tested whether these intersubunit covalent links were important to the proper VWF-binding activity of the GPIb-IX complex by characterizing the structure and VWF-binding activity of a mutant GPIb-IX complex that lacked the GPIbα-GPIbβ disulfide bonds. Results: Mutating both Cys484 and Cys485 of GPIbα to serine prevents GPIbα from forming covalent disulfide bonds with GPIbβ, while maintaining the integrity of the complex in the membrane. The mutations cause two GPIbβ subunits to form a disulfide bond between themselves. As compared to Chinese hamster ovary (CHO) cells stably expressing the wild-type GPIb-IX complex at a comparable level, CHO cells stably expressing the mutant GPIb-IX complex bind to significantly less soluble VWF in the presence of ristocetin and roll on the immobilized VWF under flow at a higher velocity. Conclusions: The disulfide bonds between GPIbα and GPIbβ are necessary for optimal GPIbα binding to VWF. The structural plasticity around the disulfide bonds may also help to shed light on the inside-out mechanism underlying GPIbβ modulation of VWF binding. © 2008 International Society on Thrombosis and Haemostasis.