Protein S secretion differences of missense mutants account for phenotypicheterogeneity

To elucidate the molecular background for the heterogeneity in protein S pl asma concentrations observed in protein S deficient individuals, the in vit ro synthesis of recombinant protein S missense mutants was investigated. Si x different naturally occurring mutations identified in the protein S gene (PROS1) of thrombosis patients were reproduced in protein S cDNA by site di rected mutagenesis, Two mutants, G441C and Y444C (group A), were associated with low total plasma concentration of protein S, Modestly low protein S w as found in families with R520G and P626L (group B) mutants. T57S and 1518M (group C), which was associated with marginally low protein S, did not seg regate with protein S deficiency in the respective families, raising doubts as to whether they were causative mutations or rare neutral variants. The 6 protein S mutants were transiently expressed in COS1 cells. The Y444C mut ant showed the lowest level of secretion (2.5%) followed by the G441C mutan t (40%). Group B demonstrated around 50% reduction in secretion, whereas gr oup C mutants showed normal secretion. Pulse-chase experiments demonstrated impaired protein S processing with intracellular degradation and decreased secretion into the culture media of group A and B mutants. Interestingly, there was a good correlation between in vitro secretion and the concentrati on of free protein S in the plasma of heterozygous carriers. These results demonstrate impaired protein S secretion to be an important mechanism under lying hereditary protein S deficiency and that variations in protein secret ion is a major determinant of the phenotypic heterogeneity observed in prot ein S deficiency. (C) 2000 by The American Society of Hematology.