Protein S Thr103Asn mutation associated with type II deficiency reproducedin vitro and functionally characterised

Protein S functions as a cofactor to activated protein C (APC) in the degra dation of Na acid FVIIIa. In protein S, the thrombin sensitive region (TSR) and the first EGF-like domain are important for expression of the APC cofa ctor activity. A naturally occurring Thr103Asn (T103N) mutation in the firs t EGF-like domain of protein S has been associated with functional (type II ) protein S deficiency. To elucidate the functional consequences of the T10 3N mutation, recombinant protein S mutant was expressed in mammalian cells and functionally characterised. The expression level of protein S T103N fro m transiently transfected COS 1 cells was equal to that of wild type protei n S. The mutant protein S and wild type protein S were also expressed in 29 3 cells after stable transfection, and the recombinant proteins purified. I n APTT- and PT-based coagulation assays, the mutant protein demonstrated ap proximately 50% lower anticoagulant activity as compared to wild type prote in S. The functional defect was further investigated in FVa- and FVIIIa-deg radation assays. The functional defect of mutant protein S was attenuated a t increasing concentrations of APC. The results demonstrate the region arou nd residue 103 of protein S to be of functional importance, possibly throug h a direct interaction with APC.