Mechanism underlying factor VII deficiency in Jewish populations with the Ala(244)Val mutation

We investigated a Sephardic Jewish patient with a mild bleeding diathesis w hose plasma levels of Factor VII coagulant activity and factor VII antigen were 7% and 9% of normal. respectively. Sequencing demonstrated homozygosit y for the Ala(244)Val mutation and the Arg(353)Gln polymorphism, which is a ssociated with a modest decrease in factor VII levels, To elucidate the mec hanism by which Ala(244)Val reduced factor VII levels in this patient, tran sient transfections were performed in COS-1 cells with wild type and mutant factor VII cDNAs and factor VII antigen levels in cell lysates and conditi oned media were measured, The secretion of the mutant protein (FVII244V) in to the media was 20% of wild type (FVIIwt), and intracellular levels of FVI I244V were 60% of FVIIwt. A construct encoding Ala(244)Val along with the A rg(353)Gln polymorphism decreased the factor VII level in the media to that observed in the patient's plasma, Pulse-chase experiments demonstrated tha t FVII244V did not accumulate intracellularly and that low levels of the ab normal protein were maintained throughout the chase. To test the hypothesis that FVII244V results in an unstable molecule, amino acids with smaller (G ly) or larger (Phe) side chains were substituted for Val(244) by site-direc ted mutagenesis, Transient transfection assays with these constructs demons trated that the side chain of amino acid 244 is crucial in maintaining a pr oper conformation of the molecule. We conclude that Ala(244)Val results in a factor VII molecule that is unstable and is probably degraded intracellul arly.