Plasma contains protein S monomers and multimers with similar direct anticoagulant activity

Summary.  Background: Protein S (PS) has activated protein C‐independent, direct anticoagulant activity (PS‐direct). We reported that both multimers and monomers of affinity‐purified PS have PS‐direct similar to that in plasma, in contrast to another report. Objective: We extended our studies to establish the molecular forms and activity of plasma PS. Methods: Novel ELISAs were developed that could detect only multimeric, not monomeric, PS because they employed the same monoclonal antibody for capture and detection. PS forms were also examined on native PAGE immunoblots. A new activity assay for PS‐direct was applied to plasma and gel‐filtered plasma fractions. Results: Plasma PS multimers were clearly demonstrated using the ELISAs; 30–60% of free plasma PS appeared to be multimeric, a proportion similar to that of affinity‐purified PS. On immunoblots, plasma PS multimers were more easily detected after gel filtration; plasma PS monomers and several apparent multimers comigrated with respective forms of affinity‐purified PS. Antigen elution profiles after gel filtration of plasma revealed at least one major peak of apparent PS multimers (40–55% of free PS appeared multimeric). Biotin‐factor Xa could bind to both plasma PS monomers and multimers. Strong plasma PS‐direct was demonstrated, and plasma PS monomers, multimers, and PS–C4b‐binding protein complexes each reconstituted PS‐depleted plasma to similar levels of PS‐direct. Conclusion: Our data are in disagreement with a report that monomeric purified PS has little PS‐direct and that only monomeric PS exists in plasma. We find that both affinity‐purified and plasma PS exist as monomers and multimers with similar PS‐direct.