The cysteine knot of platelet glycoprotein Ibβ (GPIbβ) is critical for the interaction of GPIbβ with GPIX

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Abstract
The glycoprotein Ib (GPIb) complex is composed of GPIbα covalently attached to GPIbβ and noncovalently complexed with GPIX and GPV. Patients with Bernard-Soulier syndrome demonstrate that mutations in either GPIbβ or GPIX result in an absence of platelet GPIbα. This occurs through the interaction of GPIX with GPIbβ. The precise sites of interaction of GPIbβ with GPIX are not known. To characterize the interaction of GPIbβ and GPIX, we developed an anti-GPIbβ monoclonal antibody MBC 257.4, whose epitope was in the N-terminal region of GPIbβ. N-terminal truncations of GPIbβ were expressed in mammalian cells. N-terminal truncations of GPIbβ, missing the first 14, 26, or 31 amino acids, were surface-expressed but did not enable coexpressed GPIX to be surface expressed, suggesting that the site of interaction with GPIX was modified by these deletions. GPIbβ and GPIX chimeras corresponding to predicted boundaries were used to define the sites of interaction of GPIbβ with GPIX. Replacing the N-terminal disulfide loops of GPIbβ (amino acids 1-14) with the corresponding disulfide loops of GPIX (amino acids 1-22) resulted in surface expression of coexpressed wildtype GPIX. However, when the N terminus of GPIbβ was replaced to residue 32 with the N terminus of GPIX (amino acids 1-36), GPIX did not surface express with this chimera. These results suggest that the cysteine knot region of GPIbβ in the N terminus is critical for the conformation of GPIbβ that interacts with GPIX and further suggests that a critical interaction of GPIbβ with GPIX involve residues 15 through 32 of GPIbβ.