Recombinant fibrinogen, gamma275Arg-->Cys, exhibits formation of disulfide bond with cysteine and severely impaired D:D interactions.

BACKGROUND AND OBJECTIVES

Analysis of dysfibrinogens has provided useful information aiding our understanding of molecular defects in fibrin polymerization. We have already reported impaired fibrin polymerization in a variant fibrinogen (gammaArg275Cys), the Cys being located in the D:D interface. Since this substitution occurred in a heterozygous individual, interpretation of the functional analysis was complicated. We tried to resolve this complication by synthesizing a recombinant variant fibrinogen.

METHODS

A variant gamma-chain expression plasmid was transfected into Chinese hamster ovary cells expressing normal human fibrinogen Aalpha- and Bbeta-chains. The recombinant variant fibrinogen (gamma275C) was purified using an immunoaffinity column, and we compared its structure and functions with those of normal recombinant fibrinogen (gamma275R) and plasma variant fibrinogen.

RESULTS

Mass analyses showed the existence of disulfide-linked Cys in both patient and recombinant variant fibrinogens. Functional analyses indicated that both fibrin polymerization and gamma-gamma dimer formation were markedly impaired in the variant fibrinogen. The impairments were much more pronounced in gamma275C than in plasma variant fibrinogen. In addition, scanning electron microscopic observation of fibrin clots made from gamma275C revealed less dense fibrin fiber bundles and larger fiber diameter than in those made from gamma275R, and also the existence of many aberrant fibrin fibers with tapered ends.

CONCLUSIONS

These results indicate that gammaArg275 has an important residue affecting the structure and function of the gamma-chain C-terminal domain. However, the variant D:D interface can interact with that of the normal fibrinogen existing in a heterozygous patient with dysfibrinogenemia.