The bags were then moved for 4 h to solutions containing 10 M Zn2+ in the following: 0.1 M glycine and 0.05 M NaCl, pH 2.7; TBS adjusted to pH 8; 6 M urea in TBS; or TBS containing 1 mM Mg2+ and 2 mM Ca2+. cofactor for tissue factor pathway inhibitor (TFPI) during inhibition of extrinsic FXase (8). Purified PS contains monomeric and multimeric forms, and it has been suggested that only artifactual multimeric forms of purified PS have PS-direct, measured as ability to inhibit the prothrombinase activity of FXa/FVa, by virtue of higher affinity for phospholipids (9). Yet we showed that when immunoaffinity-purified PS is separated into monomers and multimers, all forms have similar and efficient PS-direct and affinity for phospholipids on the basis of mass, comparable to the PS-direct of the PS in plasma (10). We also showed that plasma naturally contains monomeric and multimeric PS with similar PS-direct (11). Immunoaffinity-purified PS had antithrombotic activity in a baboon thrombosis model, even in the presence of blocking antibodies to APC, whereas a conventionally purified PS had weak activity (12). Immunoaffinity-purified PS from our laboratory had good PS-direct, measured as inhibition of the prothrombinase activity of FVa/FXa in the presence of saturating phospholipids, whereas conventionally purified PS from several other laboratories had poor PS-direct, which leads to skepticism regarding the existence or importance of PS-direct. Yet at least 3 labs have shown that unpurified PS in plasma has PS-direct, supporting the validity of PS-direct (13,14,15). We therefore reviewed differences in purification methods. Conventional purification often includes anion exchange chromatography with MonoQ in the presence of EDTA, followed by MonoQ with a Ca2+ gradient (16). We hypothesized that PS may contain a divalent metal ion other than Ca2+ that might be removed by MonoQ in the presence of EDTA, or by elution of barium-adsorbed vitamin K-dependent factors from plasma with high concentrations of EDTA (17). Our procedure involves elution of vitamin K-dependent factors from barium citrate pellets with ammonium sulfate rather than EDTA (18), before immunoaffinity purification that produces PS with good PS-direct (6). PS has 10 divalent metal ion binding sites that are thought to be occupied by Ca2+ (19). Most Ca2+ is located in the -carboxyglutamic acid domain at the N Peiminine terminus, where it contributes to exposure of hydrophobic residues that mediate membrane binding. Three high-affinity Ca2+ Peiminine sites are thought to be located in epidermal growth factor (EGF) domains 2C4 of PS (20, 21). Based on the crystal structure of the laminin G-1 (LG-1) domain of sex hormone binding globulin (SHBG) (22), a Ca2+ binding site was postulated to reside in the LG-1 domain of the PS SHBG-like domain (23). In the crystal structure of the homologue Gas6, the same residues of LG-1 and an additional residue in the LG-2 domain form a Ca2+ binding site that is postulated to strengthen the arrangement of the LG domain pair (24). There is scant evidence, however, that all of the metal ion binding sites in PS or in other vitamin K-dependent proteins are naturally occupied by Ca2+. We set about to identify any non-Ca2+ RAC1 divalent metal ions in PS and to correlate them with PS-direct. MATERIALS AND METHODS Proteins and reagents PS and protein C were prepared from citrated plasma by barium adsorbtion, then elution with 33% saturated ammonium sulfate (18) before further purification. For immunoaffinity purification of PS, the barium eluate was dialyzed against Tris-buffered saline (TBS; 0.05 M Tris and 0.1 M NaCl, pH 7.4), then TBS-1 mM sodium citrate. Complexes of Peiminine PS with C4b-binding protein were removed by precipitation with 3.75% (final concentration) polyethylene glycol. Crude PS was then chromatographed on a Sepharose column coupled with PS monoclonal antibody S7 and eluted with either glycine, pH 2.7, or 6 M urea. Fractions were adjusted to neutral pH and subjected to SDS-PAGE and ELISA. Selected fractions were pooled, concentrated by membrane filtration, and dialyzed twice against Hepes-buffered saline, pH 7.4 (HBS). For conventional purification, two methods were used, each beginning with barium eluate. The first was a variation of a commonly used method (16). It employed dialysis and MonoQ chromatography (quaternary amine resin from Amersham, Uppsala, Sweden) in the presence of 1 mM EDTA, then MonoQ chromatography with a Ca2+ gradient. The second method was novel and employed hexyl agarose (ProMetic, Isle of Mann, UK) chromatography of the barium eluate, with a wash of 25% saturated ammonium sulfate in HBS 1:5 and a linear gradient beginning with the wash solution and ending with HBS 1:5. A second chromatography step employed heparin Sepharose (Amersham) and.