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.
