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

The glycoprotein Ib (GPIb) complex is composed of GPIbalpha covalently attached to GPIbbeta and noncovalently complexed with GPIX and GPV. Patients with Bernard-Soulier syndrome demonstrate that mutations in either GPIbbeta or GPIX result in an absence of platelet GPIba. This occurs through the interaction of GPIX with GPIbbeta. The precise sites of interaction of GPIbbeta with GPIX are not known. To characterize the interaction of GPIbbeta and GPIX, we developed an anti-GPIbbeta monoclonal antibody MBC 257.4, whose epitope was in the N-terminal region of GPIbbeta. N-terminal truncations of GPIbbeta were ex-pressed in mammalian cells. N-terminal truncations of GPIbbeta, 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. GPIbbeta and GPIX chimeras corresponding to predicted boundaries were used to define the sites of interaction of GPIbbeta with GPIX. Replacing the N-terminal disulfide loops of GPIbbeta (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-terminal of GPIbbeta 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 GPIbbeta In the N terminus is critical for the conformation of GPIbbeta that interacts with GPIX and further suggests that a critical interaction of GPIbbeta with GPIX involve residues 15 through 32 of GPIbbeta. (C) 2002 by The American Society of Hematology.