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Consuelo Gonzlez-Manchn, Nora Butta, Gema Irun, Sonia Alonso, Matilde S. Ayuso, Roberto Parrilla
Disruption of the Cys5-Cys7 disulfide bridge in the platelet glycoprotein Ib prevents the normal maturation and surface exposure of GPIb-IX complexes
This work aimed at elucidating the molecular genetic defect in two related patients with Bernard-Soulier syndrome (BSS) phenotype. Flow cytometric analysis revealed undetectable levels of platelet glycoproteins (GP), Ib and IX, although plasma glycocalicin was detectable in both cases. The complete sequencing of GPIb, GPIb, and GPIX revealed the presence of a single point mutation, a G to A substitution, in codon 30 of GPIb, that changes Cys5 to Tyr. The parents and sibling of the patients, heterozygotes for this mutation, were asymptomatic and they all showed a reduced platelet content of GPIb and GPIX. Transient transfection of the mutant GPIb subunit failed to render surface expression of GPIb and exerted a dominant-negative effect on the surface exposure of the GPIb-IX complex. Metabolic labelling and immunoprecipitation analysis of transfected cells indicated that [5Tyr]GPIb may associate with GPIX and GPIb, but the maturation of the GPIb-IX complex is impaired. Substitution of either Cys5 or Cys7 by Ala failed to show surface expression of GPIb-IX, suggesting that the Cys5- Cys7 disulfide loop in GPIb is essential for the efficient processing and trafficking of GPIb-IX complexes toward the plasma membrane. Our findings indicate that the identified novel GPIb mutation is responsible for the BSS phenotype of the patients and provide an explanation for the molecular mechanism underlying the reduced platelet content of GPIb-IX complex in the heterozygous individuals.
Thrombosis and Haemostasis, Schattauer
Print ISSN: 0340-6245
Volume: 90, 09/2003
Pages: 456 - 464