title: Protein S multimers and monomers each have direct anticoagulant activity.
abstract: OBJECTIVE
  Plasma protein S (PS) is an essential anticoagulant that has activated protein C-independent, direct anticoagulant activity (PS-direct). It was reported that monomeric purified PS has poor PS-direct and that a subpopulation of multimeric purified PS has high PS-direct and high affinity for phospholipids. We independently examined the relative PS-direct and affinity for phospholipids of monomeric and multimeric PS and we obtained contrasting results.
  
  
  RESULTS
  Unpurified recombinant protein S (rPS) was monomeric and had PS-direct potency similar to that of both PS in plasma and multimeric affinity-purified PS, as measured in plasma assays for PS-direct and in thrombin-generation assays. Multimers of unpurified rPS were not induced by ethylenediaminetetraacetic acid (EDTA), pH 2.5, NaSCN, or barium adsorption/elution. Multimers were induced by chromatography in the presence of EDTA and thus may be concentration-dependent. In contrast to a different report, monomers, dimers, trimers, and higher-order PS forms were clearly separated in sedimentation velocity experiments and multimers were not dissociated by adding Ca(2+). Active plasma-derived and recombinant immunoaffinity-purified PS were fractionated into monomers and multimers. On a mass basis, monomers and multimers had similar specific PS-direct and ability to compete with prothrombinase components (factors Xa/Va) for limiting phospholipids. FXa ligand blotted to both monomers and multimers.
  
  
  CONCLUSIONS
  Plasma PS-direct is similar to that of affinity-purified PS and unpurified rPS. Under our conditions, monomeric and multimeric PS have similar PS-direct and ability to compete for phospholipids. Discordant earlier findings are likely due to loss of PS-direct during conventional purification procedures.
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA. heeb@scripps.edu
authors.forename: M J
authors.initials: MJ
authors.lastname: Heeb
authors.forename: P
authors.initials: P
authors.lastname: Schuck
authors.forename: X
authors.initials: X
authors.lastname: Xu
mesh_terms: D000925:Anticoagulants / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D001667:Binding, Competitive; D050716:Complement C4b-Binding Protein; D019281:Dimerization; D015951:Factor Xa / Q000378:metabolism; D006649:Histocompatibility Antigens / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D010743:Phospholipids / Q000378:metabolism; D017293:Protein S / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D014461:Ultracentrifugation
chemical_list: D000925:Anticoagulants; C055609:C4BPA protein, human; D050716:Complement C4b-Binding Protein; D006649:Histocompatibility Antigens; D008024:Ligands; D010743:Phospholipids; D017293:Protein S; D011994:Recombinant Proteins; D015951:Factor Xa