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

Abstract

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 ity for activated protein C (APC), rPST did mot show this property. rPSN and rPST bound equally to phospholipids and C4b-binding p, rotein fixed on microplate wel Is. APC bound to rPSN but mot to rPST in am 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-prothrorribinase complex was also performed. Factor Xa bound to rPSN but mot to rPST. Binding to rPSN to biotimylated factor Va in solution phase did mot 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 in the presence of rPSN but mot 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.