title: Protein S multimers are generated in vitro and affect protein S structure-function analyses.
abstract: Purified human protein S preparations contain small amounts of multimeric protein S. Protein S multimers are absent in plasma, suggesting that multimerization results from purification. Protein S multimers effectively inhibit phospholipid-dependent reactions at low phospholipid concentrations, and may therefore interfere during functional analysis of protein S. We have demonstrated that anion-exchange chromatography, as well as high ionic strength or low pH elution conditions used in immunoaffinity purification of protein S, induce protein S multimer formation. When protein S multimers were removed from protein S preparations by size-exclusion chromatography, multimers spontaneously reappeared in the protein S monomer fraction. In model systems, high phospholipid concentrations (>50 micromol/L) completely abrogate the inhibitory effect of protein S multimers on prothrombinase complex activity. In addition, C4BP does not bind to protein S multimers. Thus, at low phospholipid concentrations, addition of C4BP to purified protein S will not affect the inhibitory activity of protein S multimers. In conclusion, to avoid misinterpretations during protein S structure-function analysis due to multimers present in purified protein S preparations, we recommend studying the anticoagulant activities of protein S either in plasma, where protein S is in its unmodified natural form, or at high phospholipid concentrations in model systems with purified proteins.
authors.affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht, University Maastricht, The Netherlands.
authors.forename: Kristin M
authors.initials: KM
authors.lastname: Seré
authors.forename: George M
authors.initials: GM
authors.lastname: Willems
authors.forename: Jan
authors.initials: J
authors.lastname: Rosing
authors.forename: Tilman M
authors.initials: TM
authors.lastname: Hackeng
mesh_terms: D050716:Complement C4b-Binding Protein; D006649:Histocompatibility Antigens / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017293:Protein S / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: C055609:C4BPA protein, human; D050716:Complement C4b-Binding Protein; D006649:Histocompatibility Antigens; D046912:Multiprotein Complexes; D010743:Phospholipids; D017293:Protein S