In vitro expression demonstrates impaired secretion of the γAsn319, Asp320 deletion variant fibrinogen

Summary

The hypodysfibrinogenemia Otsu is caused by the two-residue deletion,γAsn319 and γAsp320. Analysis of plasma or purified fibrinogen from the heterozygous propositus revealed that the amount of variant γ-chain was lower than that of normal γ-chain. In order to examine the basis for this difference, we transfected Chinese hamster ovary cells and established stable cell lines that expressed both chains, γΔ/γN, only the normal chain, γN, and only the variant chain, γΔ. We measured fibrinogen concentration of confluent cultures by ELISA. We found the ratios of the concentrations in the media to the concentrations in the cell lysates of γΔ, γΔ/γN, and γN-cells were 0.42, 0.60, and 1.00, respectively. We measured the concentrations of the γΔ and γN chains by densitometric analysis of samples following separation by SDS-PAGE and found the fraction of γΔ-chains in cell lysates was always greater than the fraction in the respective culture media. We examined the kinetics of fibrinogen synthesis, assembly and secretion in pulse-chase experiments, and found that the γΔ-chain was assembled into intact fibrinogen at a rate similar to assembly of the γN-chain into normal fibrinogen, but was secreted into the medium at a slightly slower rate than normal fibrinogen. Considered together, these experiments indicate secretion of the variant fibrinogen was slightly impaired. These results suggest that the reduced level of γΔ319,320 fibrinogen in the plasma of the Otsu patient arises from modestly impaired secretion of this variant fibrinogen.

Keywords

Assembly - dysfibrinogen - pulse-chase analysis - secretion - γ-chain

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