Blood, 15 June 2002, Vol. 99, No. 12, pp. 4428-4433
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
The cysteine knot of platelet glycoprotein Ib
(GPIb) is
critical for the interaction of GPIb with GPIX
Dermot Kenny,
Patricia A. Morateck, and
Robert R. Montgomery
From the Blood Research Institute, the Blood Center of
Southeastern Wisconsin, Milwaukee; Departments of Medicine and
Pediatrics, Medical College of Wisconsin, Milwaukee; and the
Royal College of Surgeons in Dublin, Ireland.
The glycoprotein Ib (GPIb) complex is composed of GPIb
covalently attached to GPIb
and noncovalently complexed with GPIX and GPV. Patients with Bernard-Soulier 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. N-terminal truncations of
GPIb were expressed in mammalian cells. N-terminal truncations of
GPIb, 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. 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.
