The cysteine knot of platelet glycoprotein Ib (GPIb ) is critical for the interaction of GPIb with GPIX

The glycoprotein Ib (GPIb) complex is composed of GPIb covalently attached to GPIb and noncovalently complexed with GPIX and GPV. Patients with BernardSoulier syndrome demonstrate that mutations in either GPIb or GPIX result in an absence of platelet GPIb . This occurs through the interaction of GPIX with GPIb . The precise sites of interaction of GPIb with GPIX are not known. To characterize the interaction of GPIb and GPIX, we developed an anti-GPIb monoclonal antibody MBC 257.4, whose epitope was in the N-terminal region of GPIb . Nterminal truncations of GPIb were expressed in mammalian cells. N-terminal truncations of GPIb , missing the first 14, 26, or 31 amino acids, were surfaceexpressed but did not enable coexpressed GPIX to be surface expressed, suggesting that the site of interaction with GPIX was modified by these deletions. GPIb and GPIX chimeras corresponding to predicted boundaries were used to define the sites of interaction of GPIb with GPIX. Replacing the N-terminal disulfide loops of GPIb (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 GPIb 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 GPIb in the N terminus is critical for the conformation of GPIb that interacts with GPIX and further suggests that a critical interaction of GPIb with GPIX involve residues 15 through 32 of GPIb . (Blood. 2002;99:4428-4433)