In order to elucidate the role of protein C (PC) in the rat, we expressed, purified, and characterized recombinant rat PC. The purified recombinant rat PC was 70–90% two-chain (41 kDa heavy chain; 22 and 23 kDa light chain) and 10–30% single-chain (61 kDa). Amino acid analysis confirmed the presence of 10 moles of γ-carboxyglutamic acid residues per mol of protein. For comparison, plasma rat PC was purified from a barium citrate precipitate using similar method. Plasma rat PC was a two-chain form (41 kDa heavy chain; 22 kDa light chain) with no detectable single-chain nor 23 kDa light chain. For determination of the in vitro secreted species, primary cultured rat hepatocytes were incubated for 6 h with methionine-free MEM containing vitamin K1, aprotinin, and [35S]methionine. The supernatant was immunoprecipitated and analyzed by SDS-PAGE followed by autoradiography. Approximately 90% of the PC radioactivity migrated as a two-chain molecule. These results indicate that rat PC is secreted mainly as a two-chain molecule from the liver. PROTAC-activated forms of recombinant rat PC, plasma rat PC, and plasma human PC hydrolyzed the S-2366 chromogenic substrate at the same rate Recombinant rat PC was also activated by the thrombin-thrombomodulin complex at a rate similar to plasma lat PC. The anticoagulant activities of the three activated PCs were examined in rat plasma. Both recombinant and plasma rat PC prolonged the activated partial thromboplastin time in a dose-dependent manner, but plasma human PC was less effective. These results suggest that recombinant rat PC is applicable for in vivo thrombosis studies in the rat.
References
1
Dahlback B,
Fernlund P,
Stenflo J.
Inhibitors: protein C. In: Blood Coagulation.
Zwaal RF A,
Hemker HC.
(eds) Elsevier; Amsterdam: 1986: 285-306
4
Kisiel W,
Canfield WM,
Ericsson LH,
Davie EW.
Anticoagulant properties of bovine plasma protein C following activation by thrombin. Biochemistry 1977; 16: 5824-31
5
Walker FJ,
Sexton PW,
Esmon CT.
The inhibition of blood coagulation by activated protein C through the selective inactivation of activated factor V. Biochim Biophys Acta 1979; 571: 333-42
6
Marlar RA,
Kleiss AJ,
Griffin JH.
Mechanism of action of human activated protein C, a thrombin-dependent anticoagulant enzyme. Blood 1982; 59: 1067-72
9
Suzuki K,
Nishioka J,
Matsuda M,
Murayama H,
Hashimoto S.
Protein S is essential for the activated protein C-catalyzed inactivation of platelet-associated factor Va. J Biochem 1984; 96: 455-60
11
Sakata Y,
Curriden S,
Lawrence D,
Griffin JH,
Loskutoff DJ.
Activated protein C stimulates the fibrinolytic activity of cultured endothelial cells and decreases antiactivator activity. Proc Natl Acad Sci USA 1985; 82: 1121-5
13
Beckmann RJ,
Schmidt RJ,
Santerre RF,
Plutzky J,
Crabtree GR,
Long GL.
The structure and evolution of a 461 amino acid human protein C precursor and its messenger RNA, based upon the DNA sequence of cloned human liver cDNAs. Nucleic Acids Res 1985; 13: 5233-47
15
Heeb MJ,
Schwarz HP,
White T,
Lammle B,
Berrettini M,
Griffin JH.
Immunoblotting studies of the molecular forms of protein C in plasma. Thromb Res 1988; 52: 33-43
16
Greffe BS,
Manco-Johnson MJ,
Marlar RA.
Molecular forms of human protein C: comparison and distribution in human adult plasma. Thromb Haemostas 1989; 62: 902-5
17
Sugo T,
Persson U,
Stenflo J.
Protein C in bovine plasma after warfarin treatment: Purification, partial characterization, and β-hydroxyaspartic acid content. J Biol Chem 1985; 260: 10453-7
18
Zhang L,
Jhingan A,
Castellino FJ.
Role of individual γ-carboxy-glutamic acid residues of activated human protein C in defining its in vitro anticoagulant activity. Blood 1992; 80: 942-52
21
Rezaie AR,
Esmon NL,
Esmon CT.
The high affinity calcium-binding site involved in protein C activation is outside the first epidermal growth factor homology domain. J Biol Chem 1992; 267: 11701-4
22
Walker FJ.
Regulation of bovine activated protein C by protein S: the role of the cofactor protein in species specificity. Thromb Res 1981; 22: 321-7
23
Taylor FB,
Chang A,
Esmon CT,
Angelo AD,
Angelo SV-D,
Blick KE.
Protein C prevents the coagulopathic and lethal effects of Escherichia coli infusion in the baboon. J Clin Invest 1987; 79: 918-25
24
Gruber A,
Griffin JH,
Harker LA,
Hanson SR.
Inhibition of platelet-dependent thrombus formation by human activated protein C in a primate model. Blood 1989; 73: 639-42
25
Gruber A,
Hanson SR,
Kelly AB,
Yan BS,
Bang N,
Griffin JH,
Harker LA.
Inhibition of thrombus formation by activated recombinant protein C in a primate model of arterial thrombosis. Circulation 1990; 82: 578-85
26
Gruber A,
Harker LA,
Hanson SR,
Kelly AB,
Griffin JH.
Antithrombotic effects of combining activated protein C and urokinase in nonhuman primates. Circulation 1991; 84: 2454-62
28
Nawa K,
Sakano K,
Fujiwara H,
Sato Y,
Sugiyama N,
Teruuchi T,
Iwamoto M,
Marumoto Y.
Presence and function of chondroitin-4-sulfate on recombinant human soluble thrombomodulin. Biochem Biophys Res Commun 1990; 171: 729-37
33
Tanaka K,
Sato M,
Tomita Y,
Ichihara A.
Biochemical studies on liver functions in primary cultured hepatocytes of adult rats. J Biochem 1978; 84: 937-46
34
Niimi S,
Nakamura T,
Nawa K,
Ichihara A.
Hormonal regulation of translatable mRNA of tryptophan 2,3-dioxygenase in primary cultures of adult rat hepatocytes. J Biochem 1983; 94: 1697-706
35
Nawa K,
Itani T,
Ono M,
Sakano K,
Marumoto Y,
Iwamoto M.
The glycosaminoglycan of recombinant human soluble thrombomodulin affects antithrombotic activity in a rat model of tissue factor-induced disseminated intravascular coagulation. Thromb Haemostas 1992; 67: 366-70
36
Kimura M,
Kurosawa-Ohsawa K,
Takahashi M,
Koyama M,
Tanaka S,
Matsuishi T.
Purification of rabbit, rat and mouse protein C with the use of monoclonal antibody to human protein C, PC01. Thromb Res 1992; 67: 687-96
37
Grinnell BW,
Berg DT,
Walls J,
Betty Yan S.
Trans-activated expression of fully gamma-carboxylated recombinant human protein C, an antithrombotic factor. Bio/Technology 1987; 5: 1189-92
38
Betty Yan SC,
Razzano P,
Bernice Chao Y,
Walls JD,
Berg DT,
Mc Clure DB,
Grinnell BW.
Characterization and novel purification of recombinant human protein C from three mammalian cell lines. Bio/Technology 1990; 8: 655-61
39
Nelson RM,
Van Dusen WJ,
Friedman PA,
Long GL.
β-hydroxy-aspartic acid and β-hydroxyasparagine residues in recombinant human protein S are not required for anticoagulant cofactor activity or for binding to C4b-binding protein. J Biol Chem 1991; 266: 20586-9
40
Chang GT C,
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: 526-32
42
Fair DS,
Marlar RA.
Biosynthesis and secretion of factor VII, protein C. protein S, and the protein C inhibitor from a human hepatoma cell line. Blood 1986; 67: 64-70
44
Ichihara A,
Nakamura T,
Noda C,
Tanaka K.
Control of enzyme expression deduced from studies on primary cultures of hepatocytes. In: Research in isolated and cultured hepatocytes.
Guillouzo A,
Guguen-Guillouzo C.
(eds) John Libbey Eurotext; London: 1986: 187-208
45
Nawa K,
Nakamura T,
Kumatori A,
Noda C,
Ichihara A.
Glucocorticoid-dependent expression of the albumin gene in adult rat hepatocytes. J Biol Chem 1986; 261: 16883-8
46
Nakamura T,
Niimi S,
Nawa K,
Noda C,
Ichihara A,
Takagi Y,
Anai M,
Sakaki Y.
Multihormonal regulation of transcription of the tryptophan 2,3-dioxygenase gene in primary cultures of adult rat hepatocytes with special reference to the presence of a transcriptional protein mediating the action of glucocorticoids. J Biol Chem 1987; 262: 727-33
47
Mc Clure DB,
Walls JD,
Grinnell BW.
Post-translational processing events in the secretion pathway of human protein C, a complex vitamin K-dependent antithrombotic factor. J Biol Chem 1992; 267: 19710-17
