Anti-Protein S polyclonal antibody

By single strand conformational polymorphism, nucleotide sequencing and enzyme restriction, we analyzed the protein S alpha gene in 17 protein S-deficient probands and in their available family members. The relationship between genotype and phenotype was also evaluated. Twelve different sequence variations were identified in 17 probands. Ten were putative causal mutations distributed in 16 probands: 4 were nonsense, 5 missense and one a splice site mutation. In most families in which a mutation was identified, more than one phenotype: of PS deficiency was present. The same splice site mutation (intron j G-A, exon 10+5) was associated with type I deficiency in one family and with type I/III in another unrelated family. A phenotypic discrepancy was also observed for the Arg474Pro, Gly597Asp and Arg410stop mutations. Glu26Ala, previously reported in kindreds with type I deficiencies, was found in association with I, II and LU phenotypes in four unrelated kindreds. Phenotypic analysis of protein S deficiency is poorly related to the underlying genetic defect.

To elucidate the molecular background for the heterogeneity in protein S plasma concentrations observed in protein S deficient individuals, the in vitro synthesis of recombinant protein S missense mutants was investigated. Six different naturally occurring mutations identified in the protein S gene (PROS1) of thrombosis patients were reproduced in protein S cDNA by site directed mutagenesis, Two mutants, G441C and Y444C (group A), were associated with low total plasma concentration of protein S, Modestly low protein S was 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 segregate 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 mutant showed the lowest level of secretion (2.5%) followed by the G441C mutant (40%). Group B demonstrated around 50% reduction in secretion, whereas group 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 concentration of free protein S in the plasma of heterozygous carriers. These results demonstrate impaired protein S secretion to be an important mechanism underlying hereditary protein S deficiency and that variations in protein secretion is a major determinant of the phenotypic heterogeneity observed in protein S deficiency. (C) 2000 by The American Society of Hematology.