In vitro characterisation of two naturally occurring mutations in the thrombin-sensitive region of anticoagulant protein S

The molecular consequences of two naturally occurring mutations in the thro mbin-sensitive region of protein S were investigated using a combination of recombinant protein expression, functional analysis and molecular modellin g. Both mutations (R49H and R70S) have been found in thrombosis patients di agnosed as having type I protein S deficiency. Molecular modelling analysis suggested the R49H substitution not to disrupt the structure of thrombin-s ensitive region, whereas the R70S substitution could affect the 3D structur e mildly. To elucidate the molecular consequences of these substitutions ex perimentally, site directed mutagenesis of protein S cDNA and expression in mammalian cells created the two mutants. The secretion profiles and functi onal anticoagulant activities of the protein S mutants were characterised. Secretion of the R49H mutant was similar to that of wild type protein S, wh ereas the R70S mutant showed moderately decreased expression. Neither of th e mutants showed any major functional defects as cofactors to activated pro tein C (APC) in an APTT-based assay or in degradation of factor Va. However , both mutants demonstrated decreased activity in a factor VIIIa degradatio n assay, which in addition to APC and protein S also included factor V as s ynergistic APC cofactor. In conclusion, the R49H substitution did not produ ce a quantitative abnormality in vitro, raising doubts as to whether it cau sed the type I deficiency. In contrast, the experimental data obtained for the R70S mutant agrees well with the observed type I deficiency. Our study illustrates that in vitro experimental characterisation together with compu ter-based structural analysis are useful tools in the analysis of the relat ionship between naturally occurring mutations and clinical phenotypes.