Zn2+‐containing protein S inhibits extrinsic factor X‐activating complex independently of tissue factor pathway inhibitor

Background: Protein S (PS) has direct anticoagulant activity, independently of activated protein C (APC). The mechanisms underlying this activity remain unclear, because PS preparations differ in activity, giving rise to conflicting results. Some purification procedures result in loss of intramolecular Zn2+, which is essential for inhibition of prothrombinase. Objective: To investigate the inhibition of extrinsic factor (F)Xase by Zn2+‐containing PS. Methods: Purified component extrinsic FXase assays were used to determine FXa generation in the presence and absence of PS and/or tissue factor pathway inhibitor (TFPI). Binding assays, immunoblots and thrombin generation assays in plasma supported the FXase data. Results: Zn2+‐containing PS potently inhibited extrinsic FXase in the presence of saturating phospholipids, independently of TFPI, whereas inhibition of extrinsic FXase by Zn2+‐deficient PS required TFPI. Immunoblots for FXa and functional assays showed that Zn2+‐containing PS inhibited primarily the quantity of FXa formed by tissue factor (TF)–FVIIa, rather than FXa amidolytic activity. Zn2+‐containing PS, but not Zn2+‐deficient PS, bound to TF with high affinity (Kd app = 41 nm) and targeted TF function. Binding of PS to FVIIa was negligible, whereas PS showed appreciable binding to FX. Increasing FX concentrations 10‐fold reduced PS inhibition five‐fold, suggesting that PS inhibition of FXase is FX‐dependent. PS also exhibited TFPI‐independent and APC‐independent anticoagulant activity during TF‐initiated thrombin generation in plasma. Conclusions: PS that retains native Zn2+ also retains anticoagulant functions independently of APC and TFPI. Inhibition of extrinsic FXase by PS at saturating levels of phospholipids depends on PS retention of intramolecular Zn2+, interaction with FX, and, particularly, interaction with TF.