A Novel Missense Mutation Shows that GPIbβ Has a Dual Role in Controlling the Processing and Stability of the Platelet GPIb-IX Adhesion Receptor

Glycoprotein (GP) Ibα is a major adhesive receptor of platelets, surface expressed as part of the GPIb-IX-V complex. However, important questions about how the four gene products (Ibα, Ibβ, IX, and V) composing this complex are processed remain. A deficiency of or nonfunctioning GPIb-IX-V is characteristic of the Bernard-Soulier syndrome (BSS), an inherited bleeding disease. We now report a BSS variant whose platelets have little or no GIbβ or GPIX, but where residual GPIbα was selectively located in flow cytometry by monoclonal antibodies (WM23 and Bx-1) recognizing denatured epitopes. Whereas WM23 immunoprecipitated GPIbα (130 kDa), GPIX, and GPIbβ from control platelets, a single surface protein of ≈66 kDa was obtained for the patient. DNA sequencing revealed a homozygous Asn64 → Thr substitution in the GPIbβ from the patient. This substitution modified a conserved residue in the COOH-terminal region flanking the single-copy leucine-rich domain of GPIbβ. When GPIbβ64Thr was coexpressed in a stable CHO cell line with wild-type GPIbα and GPIX, flow cytometry and confocal microscopy failed to show GPIb-IX complexes at the cell surface. Intracellular GPIbα and GPIbβ were detected and largely confined to the endoplasmic reticulum, and little GPIX was seen. GPIbα was immunoprecipitated as a 66−70 kDa protein in 35S metabolic studies and lacked O-glycosidic side chains. Also, it was not disulfide bound to the mutated GPIbβ. Thus, a single amino acid substitution in the extracellular domain of GPIbβ can affect both the maturation of GPIbα and GPIX stability. GPIbβ has a pivotal role in regulating GPIb-IX-V biosynthesis.