Rabbit Plasma, unlike Its Human Counterpart, Contains no Complex between Protein S and C4b-Binding Protein

Summary In human plasma, the anticoagulant vitamin K-dependent protein S exists in two molecular forms, as free protein and complexed to C4b- binding protein (C4BP), a complement regulatory protein. It has been suggested that rabbit plasma also contains two forms of protein S and that the interaction between protein S and C4BP m rabbits can be modulated by synthetic peptides corresponding to a sequence (residues 605-614) in the carboxy-lerminal part of protein S. In this report, we provide itsulls which challenge the conclusion that rabbit plasma contains the complexed form of protein S. The two forms of protein S in human plasma were separated by gel filtration chromatography on Sephacryl S-300 and the presence of protein S in the various fractions analyzed by Western blotting using a monoclonal antibody (HPS 21) directed against the γ-carboxyglutamic acid rich module of human protein S. This antibody, which was found to cross-react with rabbit protein S on Western blotting, was used in affinity purification of protein S from rabbit plasma as well as of recombinant rabbit protein S. HPS 21 specifically recognized protein S in rabbit plasma and did not cross-react with the other vitamin K-depeudenl plasma proteins. To elucidate whether rabbit plasma contained two forms of protein S, rabbit plasma was subjected to gelfiltration chromatography followed by Western blotting of the fractions with monoclonal antibody HPS 21. Protein S was found only in fractions eluting at a position corresponding to that of free protein S. A radiolabelled trace amount of recombinant rabbit protein S added to rabbit plasma chromatographed as free protein S and no high molecular weight form corresponding to a C4BP-protein S complex was detected. Rabbit protein S had the capacity to bind human C4BP and the addition of human C4BP to rabbit plasma changed the elution profile, of rabbit plasma protein S. After the addition of human C4BP, rabbit plasma protein S quantitatively eluted as a high molecular weight complex, suggesting that all the protein S in rabbit plasma was bound to human C4BP. The anticoagulant activity of human protein S is modulated by the complex formation with C4BP. Our results demonstrate that this function of C4BP and the protein S-C4BP complex formation has not been conserved throughout the evolution even though protein S has a preserved C4BP binding site.