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.To investigate the inhibition of extrinsic factor (F)Xase by Zn²(+) -containing PS.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.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 (K(dapp) = 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.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.