Published online 26 January 2008
Haematologica, Vol 93, Issue 2, 224-231 doi:10.3324/haematol.11868
Copyright © 2008 by Ferrata Storti Foundation
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Hemostasis

Manipulating the quality control pathway in transfected cells: low temperature allows rescue of secretion-defective fibrinogen mutants

Dung Vu1, Corinne Di Sanza1, Marguerite Neerman-Arbez1,2,

1 Department of Genetic Medicine and Development, University Medical Center, Geneva;
2 Division of Angiology and Hemostasis, University Hospital, Geneva, Switzerland

Correspondence: Marguerite Neerman-Arbez, 1 rue Michel Servet, CH-1211 Geneva, Switzerland. E-mail: marguerite.arbez{at}medecine.unige.ch

Background: Congenital afibrinogenemia is characterized by the absence of fibrinogen, a hexamer composed of two copies of three polypeptides, A{alpha}. Bβ and {gamma}. The disease is caused by mutations in one of the three fibrinogen-encoding genes, FGA, FGB and FGG. Among these, several mutations have been reported to specifically impair fibrinogen secretion. We previously showed that secretion-defective fibrinogen mutants are retained in a pre-Golgi compartment and demonstrated the importance of the homologous βC and {gamma}C domains in secretion. Here our aim was to restore the secretion of these mutants and study the properties of the rescued mutant molecules.

Design and Methods: COS-7 cells were transfected and incubated with chemical chaperones or at low temperature. Clotting assays and plasmin digestion studies were performed to characterize secreted fibrinogen molecules.

Results: The secretion defect of two missense mutants but not that of late-truncating mutants could be partially corrected by incubating cells at 27°C. By contrast, exposure of cells to chemical chaperones i.e. 4-phenylbutyrate, dimethyl sulfoxide or trimethylamine N-oxide had no effect. The mutants rescued at 27°C were incorporated into fibrin clots and formed factor XIII-mediated {gamma}-{gamma} dimers in contrast to the dysfibrinogenemia Vlissingen/Frankfurt IV mutant, a negative control for these assays. However, plasmin digestion analyses revealed aberrant patterns for the mutants compared to normal fibrinogen.

Conclusions: Low temperature can restore the secretion of a subset of mutant fibrinogen molecules demonstrating that therapeutic manipulation of the quality control pathway is feasible for afibrinogenemia even though functional assays suggested a non-native conformation for the mutant molecules analyzed.

Key words: fibrinogen, afibrinogenemia, secretion, chemical chaperones, protein quality control, fibrinogen-related domain.




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