title: Reevaluation of total, free, and bound protein S and C4b-binding protein levels in plasma anticoagulated with citrate or hirudin.
abstract: The levels of total, free, and bound protein S and C4BP were determined using enzyme-linked immunosorbent assays (ELISAs) in plasma samples (8 males and 8 females) that were individually subjected to immunoadsorption studies in which "free protein S" was defined as that not adsorbed by anti-C4BP antibody-Sepharose column and "free C4BP" as that not adsorbed by anti-protein S antibody-Sepharose. Bound species were calculated as the difference between total and free species. Free protein S (131 nmol/L) averaged 38% of total protein S (346 nmol/L) and free C4BP (37 nmol/L) averaged 14% of total C4BP (264 nmol/L) in plasma. There was an excellent correlation between bound protein S and bound C4BP with 1:1 molar stoichiometry and a good correlation between free protein S antigen and protein S anticoagulant activity. It appears that free protein S is a necessary consequence of the molar excess of protein S over C4BP. C4BP beta chain antigen levels, measured using a new ELISA, averaged 218 nmol/L, a value indistinguishable from the molar concentrations of bound protein S (215 nmol/L) and bound C4BP (227 nmol/L). The free C4BP beta chain antigen was only 4 nmol/L compared with 131 nmol/L free protein S. These results suggest that free C4BP in plasma predominantly lacks the beta chain, that almost all C4BP capable of binding protein S is associated with protein S, and that the plasma levels of oligomeric forms of C4BP containing a beta chain (alpha 7 beta and alpha 6 beta) that bind protein S directly and stoichiometrically regulate free protein S levels.
authors.affiliation: Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037.
authors.forename: J H
authors.initials: JH
authors.lastname: Griffin
authors.forename: A
authors.initials: A
authors.lastname: Gruber
authors.forename: J A
authors.initials: JA
authors.lastname: Fernández
mesh_terms: D000925:Anticoagulants / Q000097:blood*; D002352:Carrier Proteins / Q000097:blood*; D002951:Citrates / Q000097:blood*; D019343:Citric Acid; D003169:Complement Inactivator Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006023:Glycoproteins / Q000097:blood*; D006629:Hirudins / Q000097:blood*; D006801:Humans; D007163:Immunosorbent Techniques; D008297:Male; D008970:Molecular Weight; D010314:Partial Thromboplastin Time; D011485:Protein Binding; D017293:Protein S; D012016:Reference Values
chemical_list: D000925:Anticoagulants; D002352:Carrier Proteins; D002951:Citrates; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D006629:Hirudins; D017293:Protein S; D019343:Citric Acid