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

AUTOR(ES)

Heeb, Mary J.

FONTE

The Federation of American Societies for Experimental Biology

RESUMO

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.

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