Characterization of dysfunctional protein S-Tokushima (K155-->E) in relation to the molecular interactions required...

Protein S is a vitamin K-dependent plasma protein that acts as a cofactor of activated protein C (APC) and as an inhibitor of the prothrombinase complex. We previously reported that protein S-Tokushima, found in a patient with heterozygous protein S deficiency and severe venous thrombotic disease, has a substitution of Glu for Lys-155 in the second epidermal growth factor (EGF)-like domain of protein S. In the present study, we characterized the role of protein S, which is required for the expression of the APC cofactor activity and for the inhibition of the prothrombinase complex activity, using recombinant normal protein S (rPS-N) and protein S-Tokushima (rPS-T), expressed in BHK cells. rPS-N as well as plasma protein S showed APC cofactor activity, but rPS-T did not. Both rPS-N and rPS-T equally bound to phospholipids (Cephalin) and C4b-binding protein fixed to microwells. APC bound to rPS-N and plasma protein S, but did not bind to rPS-T. Although rPS-N as well as plasma protein S inhibited the platelet prothrombinase complex activity, rPS-T did not. Factor Xa bound to rPS-N but not to rPST. Binding of rPS-N to biotinylated factor Va in solution phase did not differ significantly from that of rPS-T. The dysfunction of protein S-Tokushima is suggested to be mainly caused by a lack of interaction with APC required for the expression of the APC cofactor activity and by a lack of interaction with factor Xa required for the inhibition of the prothrombinase complex activity.