Plasma protein S contains zinc essential for efficient activated protein C-independent anticoagulant activity and binding to factor Xa, but not for efficient binding to tissue factor pathway inhibitor

摘要

Protein S (PS) is a cofactor for activated protein C (APC), which inactivates coagulation factors (F) Va and VIIIa. Deficiency of protein C or PS is associated with risk of thrombosis. We found that PS also has APC-independent anticoagulant activity (PS-direct) and directly inhibits thrombin generated by FXa/FVa (prothrombinase complex). Here we report that PS contains Zn2+ that is required for PS-direct and that is lost during certain purification procedures. Immunoaffinity-purified PS contained 1.4 ± 0.6 Zn2+/mol, whereas MonoQ-purified and commercial PS contained 0.15 ± 0.15 Zn2+/mol. This may explain the controversy regarding the validity of PS-direct. Zn2+ content correlated positively with PS-direct in prothrombinase assays and clotting assays, but APC-cofactor activity of PS was independent of Zn2+ content. PS-direct and Zn2+ were restored to inactive PS under mildly denaturing conditions. Conversely, o-phenanthroline reversibly impaired the PS-direct of active PS. Zn2+-containing PS bound FXa more efficiently (Kdapp=9.3 nM) than Zn2+-deficient PS (Kdapp=110 nM). PS bound TFPI efficiently, independently of Zn2+ content (Kdapp=21 nM). Antibodies that block PS-direct preferentially recognized Zn2+-containing PS, suggesting conformation differences at or near the interface of 2 laminin G-like domains near the PS C terminus. Thus, Zn2+ is required for PS-direct and efficient FXa binding and may play a role in stabilizing PS conformation.—Heeb, M. J., Prashun, D., Griffin, J. H., Bouma, B. N. Plasma protein S contains zinc essential for efficient activated protein C-independent anticoagulant activity and binding to factor Xa, but not for efficient binding to tissue factor pathway inhibitor.