Rabbit plasma, unlike its human counterpart, contains no complex between protein S and C4b-binding protein

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 in rabbits can be modulated by synthetic peptides corresponding to a sequence (residues 605-614) in the carboxy-terminal part of protein S. In this report, we provide results 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 gamma-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-dependent plasma proteins. To elucidate whether rabbit plasma contained two forms of protein S, rabbit plasma was subjected to gel-filtration 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.(ABSTRACT TRUNCATED AT 250 WORDS)