Molecular mechanism of the dysfunction of protein S-Tokushima (Lys(155)->Glu) for the regulation of the blood coagulation system

The congenital abnormal protein S-Tokushima has Glu substituted for Lys(155) in the second epidermal growth factor domain of the protein S molecule (Hayashi T., Nishioka J., Shigekiyo, T. Saito; S. and Suzuki, K. (1994) Blood 83, 683-690). To elucidate the molecular mechanism of the dysfunction of the protein S-Tokushima, a comparative evaluation between the molecular interaction of the abnormal protein S and that of normal protein S with other clotting factors was carried out using recombinant normal protein S (rPSN) and protein S-Tokushima (rPST) expressed in baby hamster kidney cells. While rPSN and plasma protein S exhibited cofactor activity for activated protein C (APC), rPST did not show this property. rPSN and rPST bound equally to phospholipids and C4b-binding protein fixed on microplate wells. APC bound to rPSN but not to rPST in an assay using immobilized monoclonal anti-protein S antibody. On the other hand, rPSN and plasma protein S inhibited the activity of prothrombinase complex composed of factor Xa and thrombin-stimulated platelets, whereas rPST lacked this inhibitory effect. Assessment of the mechanism by which rPST lacks inhibitory activity on the platelet-prothrombinase complex was also performed. Factor Xa bound to rPSN but not to rPST. Binding to rPSN to biotinylated factor Va in solution phase did not differ significantly from that of rPST. Binding of prothrombin to factor Va in solution phase was not inhibited either by rPSN or rPST. Binding of 4-amidinophenylmethanesulfonyl-factor Xa to factor Va in solution phase increased in the presence of rPSN but not in that of rPST. These findings suggest that the dysfunction of protein S-Tokushima occurs because it fails to interact with APC and factor Xa. This molecular interaction is required for the expression of the APC cofactor activity and for the inhibition of the prothrombinase complex activity.