Exploring the intracellular fate of coagulation factor V carrying the R2074C mutation

Severe factor V (FV) deficiency is a rare coagulation disorder, characterized by very low or unmeasurable plasma levels of functional and immunoreactive FV, associated with a hemorrhagic phenotype of variable severity. It is transmitted as an autosomal recessive trait with a prevalence of about 1 in 1 million and, among rare inherited coagulopathies, is the least characterized from the molecular point of view. So far, only 3 missense mutations (C472G, V1813M, and R2074C) were characterized by in-vitro expression by our group, demonstrating in all cases an impaired secretion associated with intracellular degradation. Among these mutations, R2074C was associated with the lowest intracellular antigen levels, suggesting an efficient intracellular degradation of the unsecreted protein. Here we report a further characterization of the degradation pathway, performed by studying the effect of different inhibitors of protein degradation (NH4Cl, leupeptin, lactacystin, ALLN, brefeldin A) on recombinant FV expressed in COS-1 cells. The obtained results suggested that FV-R2074C is subjected to intracellular degradation by the ubiquitin-proteasome pathway.