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Blood, 1 May 2001, Vol. 97, No. 9, pp. 2640-2647
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Competition between normal [674C] and mutant [674R]GPIIb
subunits: role of the molecular chaperone BiP in the processing of
GPIIb-IIIa complexes
Elena G. Arias-Salgado,
Nora Butta,
Consuelo González-Manchón,
Susana Larrucea,
Matilde S. Ayuso, and
Roberto Parrilla
From the Department of Pathophysiology and Human
Molecular Genetics, Centro de Investigaciones Biológicas (CSIC),
Madrid, Spain.
This work aimed at investigating the function of the [C674R]
mutation in GPIIb that disrupts the intramolecular 674 to 687 disulfide
bridge. Individuals heterozygous for this mutation show a platelet
GPIIb-IIIa content approximately 30% of normal controls, which is less
than expected from one normal functioning allele. Coexpression of
normal [674C]GPIIb and mutant [674R]GPIIb with normal
GPIIIa produced a [674R]GPIIb concentration-dependent inhibition of surface exposure of GPIIb-IIIa complexes in Chinese hamster ovary
(CHO) cells, suggesting that [674R]GPIIb interferes with the
association and/or intracellular trafficking of normal subunits. Mutation of either 674C or 687C had similar effects in reducing the
surface exposure of GPIIb-IIIa. However, substitution of 674C for A
produced a much lesser inhibition than R, suggesting that a
positive-charged residue at that position renders a less efficient subunit conformation. The mutant [674R]GPIIb but not normal GPIIb was found associated with the endoplasmic reticulum chaperone BiP in
transiently transfected CHO cells. BiP was also found associated with
[674R]GPIIb-IIIa heterodimers, but not with normal GPIIIa or
normal heterodimers. Overexpression of BiP did not increase the surface
exposure of [674R]GPIIb-IIIa complexes, indicating that its
availability was not a limiting step. Platelets from the thrombasthenic
patient expressing [674R]GPIIb-IIIa were found to bind soluble
fibrinogen in response to physiologic agonists or dithiothreitol
treatment. Thus, the [674R]GPIIb mutation leads to a retardation
of the secretory pathway, most likely related to its binding to the
molecular chaperone BiP, with the result of a defective number of
functional GPIIb-IIIa receptors in the cell surface.
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