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Glycoprotein (GP) Ibalpha 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 (Ibalpha, Ibbeta, 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 GIbbeta or GPIX, but where residual GPIbalpha was selectively located in flow cytometry by monoclonal antibodies (WM23 and Bx-1) recognizing denatured epitopes. Whereas WM23 immunoprecipitated GPIbalpha (130 kDa), GPIX, and GPIbbeta from control platelets, a single surface protein of approximately 66 kDa was obtained for the patient. DNA sequencing revealed a homozygous Asn(64) --> Thr substitution in the GPIbbeta from the patient. This substitution modified a conserved residue in the COOH-terminal region flanking the single-copy leucine-rich domain of GPIbbeta. When GPIbbeta64Thr was coexpressed in a stable CHO cell line with wild-type GPIbalpha and GPIX, flow cytometry and confocal microscopy failed to show GPIb-IX complexes at the cell surface. Intracellular GPIbalpha and GPIbbeta were detected and largely confined to the endoplasmic reticulum, and little GPIX was seen. GPIbalpha was immunoprecipitated as a 66-70 kDa protein in (35)S metabolic studies and lacked O-glycosidic side chains. Also, it was not disulfide bound to the mutated GPIbbeta. Thus, a single amino acid substitution in the extracellular domain of GPIbbeta can affect both the maturation of GPIbalpha and GPIX stability. GPIbbeta has a pivotal role in regulating GPIb-IX-V biosynthesis.