Reevaluation of total, free, and bound protein S and C4b-binding protein levels in plasma anticoagulated with citrate or hirudin

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 β 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 β 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 β 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 β chain (α₇β and α₆β) that bind protein S directly and stoichiometrically regulate free protein S levels.