In vitro expression studies and immunofluorescence microscopy analysis of two naturally occuring mutations on Factor X (FX) gene (G94R and D95E)

Factor X (FX) deficiency is one of the most severe among recessively inherited coagulation disorders. Two mutations, G94R and D95E, located in the EGF2 domain of FX, both in homozygous state, were previously identified in 2 Iranian brothers affected by FX deficiency with 35% FX : C activity measured by the RVV test, undetectable PT- and PTT-based FX : C associated with low chromogenic activity (8%) and 3% FX : Ag levels. We investigated the role of each single mutation alone and together by in vitro expression studies and immunofluorescence microscopy staining analysis on wild type and mutant recombinants FX (FXWT, FX95E, FX94R and FX94R +95E) obtained by transient transfections. After 72 hours of transfection the procoagulant activity of FXWT and mutants were measured on the conditioned media, whereas FX : Ag levels were measured in both cell lysates and conditioned media. Results: Procoagulant activity of FXWT and FX95E were normal, whereas FX94R and FX94R +95E showed no procoagulant activity. FX : Ag in cell lysates transfected by mutant constructs FX95E, FX94R and FX94R +95E were ca 75%, 50% and 40%. The conditioned media gave values of 85%, 5% and 4%, respectively compared to FXWT. Immunofluorescence microscopy staining showed that FXWT and FX95E were mostly localized in the perinuclear area, whereas FX94R and FX94R +95E were present diffusely throughout the cytoplasm, with no perinuclear enhancement. Conclusions: The G94R mutation is probably responsible for intracellular degradation of FX leading to a secretion defect, while the D95E had no significant effect on synthesis, secretion and procoagulant activity of FX. On the basis of the discrepancy observed in patients’ plasma between RVV-, PT- and PTT-based assays, the hypothesis of impaired FX94R activation by FIXa-FVIIIa or FVIIa-TF complexes is purported.