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

Abstract

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). Biotinfactor 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.

TAM receptors, Gas6, and protein S: roles in inflammation and hemostasis

“…91 Similar high-molecularweight multimeres of protein S have been observed in plasma and reported to possess equal anticoagulant properties as the monomers. 92 Membranes that contain phosphatidylserine serve as a scaffold for the auto-oxidation of Cys residues in protein S, which promotes the oligomerization of protein S required for Mer-dependent apoptotic cell clearance. 73 This oligomerization may also provide a mechanism that allows the abundant protein S to activate Mer when necessary and not constitutively.…”

The Kunitz-3 domain of TFPI-α is required for protein S–dependent enhancement of factor Xa inhibition

“…29 Whether multimers of PS are present in plasma or are generated during PS purification is controversial. [29][30][31][32][33] More recently, it was shown that PS enhances the inhibition of FXa by TFPI-␣ and that this action of PS is apparent with monomeric PS. 12,13,34 TFPI-␣ regulates blood coagulation by directly inhibiting FXa through its K2 domain and, in a FXa-dependent fashion, inhibiting FVIIa/TF through its K1 domain.…”

The Thrombin-sensitive Region of Protein S Mediates Phospholipid-dependent Interaction with Factor Xa

“…However, they observed that protein S multimers were not observed in plasma and so concluded that these forms of protein S are unlikely to contribute to protein S direct in plasma. Whether protein S multimers are present in plasma or generated during purification is also a matter of debate (27)(28)(29)(30). In our studies, we have used commercial protein S preparations that essentially have only protein S monomers.…”