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.
References
1
Esmon CT.
The role of protein C and thrombomodulin in the regulation of blood coagulation. J Biol Chem 1989; 264: 4743-6
2
Dahlback B,
Stenflo J.
The protein C anticoagulant system. In: Stamatoyannopoulos G
Nienhuis AW,
Majerus PW,
Varmus H.
(eds)
The molecular basis of blood diseases. Philadelphia W. B. Saunders; 1993: 599-628
3
Kane WH,
Davie EW.
Blood coagulation factors V and VIII: structural and functional similarities and their relationship to the hemorrhagic and thrombotic disorders. Blood 1988; 71: 539-55
4
Mann KG,
Jenny RJ,
Krishnaswamy S.
Cofactor proteins in the assembly and expression of blood clotting enzyme complexes. Ann Rev Biochem 1988; 57: 915-56
8
Fair DS,
Marlar RA.
Biosynthesis and secretion of factor VIII, protein C, protein S, and the protein C inhibitor from a human hepatoma cell line. Blood 1986; 67: 64-70
9
Stem D,
Brett J,
Harris K,
Nawroth P.
Participation of endothelial cells in the protein C protein S anticoagulant pathway: the synthesis and release of protein S. J Cell Biol 1986; 102: 1971-8
11
Malm J,
Xtihua He,
Bjartell A,
Shen Lei,
Abrahamsson PA,
Dahlback B.
Synthesis of vitamin K-dependent protein S by leydig cells of human testis Thromb Haemostas 1993; 69: 933
12
Dahlbdck B.
Protein S and C4b-biudrng protein. Components involved in the regulation of the protein C anticoagulant system. Thromb Haemostas 1991; 66: 49-61
13
Dahlback B,
Stenflo J.
High molecular weight complex in human plasma between vitamin K-dependent protein S and complement component C4b- binding protein. Proc Natl Acad Sci USA 1981; 78: 2512-6
14
Dahlback B.
Interaction between vitamin K-dependent protein S and the complement protein, C4b-binding protein: A link between coagulation and the complement system. Semin Thromb Haemostas 1984; 10: 139-18
15
Walker FJ.
The regulation of activated protein C by a new protein: a possible function for bovine protein S. Semin Thromb Haemostas 1980; 255: 5521-4
18
Manfioletti G,
Brancolini C,
Avanzi G,
Schneider C.
The protein encoded by a growth arrest-specific gene (Gas6) is a new member of the vitamin K- dependent proteins related to protein S, a negative coregulator in the blood coagulation cascade. Mol Cell Biol 1993; 13: 4976-85
21
Hardig Y,
Rezaie A,
Dahlback B.
High affinity binding of human vitamin K-dependent protein S to a truncated recombinant (3-chain of C4b-binding protein. J Biol Chem 1993; 268: 3033-6
22
Dahlback B,
Smith CA,
Miiller-Eberhard HJ.
Visualization of human C4b- binding protein and its complexes with vitamin K-dependent protein S and complement protein C4b. Proc Natl Acad Sci USA 1984; 80: 3461-5
23
Fujita T,
Kamato T,
Tamura N.
Characterization of functional properties of C4b-binding protein by monoclonal antibodies. J Immunol 1985; 134: 3320-4
24
Chung LP,
Reid KBM.
Structural and functional studies on C4b-binding protein, a regulatory component of the human complement system. Biosci Rep 1985; 855-65
25
Hessing M,
Kanter D,
Takeya H,
van’t Veer C,
Hackgeng TM,
Iwanaga S,
Bouma BN.
The region Ser333-Arg356 of the a-chain of human C4b-binding protein is involved in the binding of complement C4b. FEBS Lett 1993; 317: 228-32
26
Walker FJ.
Characterization of a synthetic peptide that inhibits the interaction between protein S and C4b-binding protein. J Biol Chem 1989; 264: 17645-8
28
Fernandez JA,
Heeb MJ,
Griffin JH.
Identification of residues 413-433 of plasma protein S as essential for binding to C4b-binding protein. J Biol Chem 1993; 268: 16788-94
29
Dahlbäck B,
Frohm B,
Nelsestuen G.
High affinity interaction between C4b-binding protein and vitamin K-dependent protein S in the presence of calcium. J Biol Chem 1990; 265: 16082-7
31
Walker FJ.
Identification of a new protein involved in the regulation of the anticoagulant activity of activated protein C: protein S binding protein. J Biol Chem 1986; 261: 10941-4
32
Weinstein RE,
Walker FJ.
Enhancement of rabbit protein S anticoagulant cofactor activity in vivo by modulation of the protein S C4b-binding protein interaction. J Clin Invest 1990; 86: 1928-35
33
Xuhua He,
Dahlback B.
Molecular cloning, expression and functional characterization of rabbit anticoagulant vitamin K-dependent protein S. Eur J Biochem 1993; 217: 857-65
36
Malm J,
Persson U,
Dahlback B.
Inhibition of human vitamin K-dependent protein S cofactor activity by a monoclonal antibody specific for a Ca2+-de- pendent epitope. Eur J Biochem 1987; 165: 39-45
37
Dahlback B,
Hildebrand B,
Malm J.
Characterization of functionally important domains in human vitamin K-dependent protein S using monoclonal antibodies. J Biol Chem 1990; 265: 8127-35
38
Malm J,
Cohen E,
Dackowski W,
Dahlback D,
Wydro R.
Expression of completely γ-carboxylated recombinant human vitamin K-dependent protein S with full biological activity. Eur J Biochem 1990; 187: 737-43
42
Taylor Jr FB,
Chang A,
Esmon CT,
D’Angelo AS,
Vigano-D’Angelo S,
Blick KE.
Protein C prevents the coagulopathic and lethal effects of Escherichia Coli infusion in the baboon. J Clin Invest 1987; 79: 918-25
43
Chang GTG,
Ploos van Amstel HK,
Hessing M,
Reitsma PH,
Bertina RM,
Bouma BN.
Expression and characterization of recombinant human protein S in heterologous cells-studies of the interaction of amino acid residues Leu-608 to Glu-612 with human C4b-binding protein. Thromb Haemostas 1992; 67: 526-532
44
Them A,
Zoller B,
Dahlback B.
The lack of complexes between protein S and C4b-binding protein in bovine plasma is explained by the primary structure of the (3-chain of bovine C4b-binding protein. Thromb Haemostas 1993; 69: 932
