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Human Plasma Protein C: ISOLATION, CHARACTERIZATION, and MECHANISM of ACTIVATION by Α-THROMBIN

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Human Plasma Protein C: ISOLATION, CHARACTERIZATION, and MECHANISM of ACTIVATION by Α-THROMBIN Human Plasma Protein C: ISOLATION, CHARACTERIZATION, AND MECHANISM OF ACTIVATION BY α-THROMBIN Walter Kisiel J Clin Invest. 1979;64(3):761-769. https://doi.org/10.1172/JCI109521. Protein C is a vitamin K-dependent protein, which exists in bovine plasma as a precursor of a serine protease. In this study, protein C was isolated to homogeneity from human plasma by barium citrate adsorption and elution, ammonium sulfate fractionation, DEAE-Sephadex chromatography, dextran sulfate agarose chromatography, and preparative polyacrylamide gel electrophoresis. Human protein C (Mr = 62,000) contains 23% carbohydrate and is composed of a light chain (Mr = 21,000) and a heavy chain (Mr = 41,000) held together by a disulfide bond(s). The light chain has an amino-terminal sequence of Ala-Asn-Ser-Phe-Leu- and the heavy chain has an aminoterminal sequence ofA sp-Pro-Glu- Asp-Gln. The residues that are identical to bovine protein C are underlined. Incubation of human protein C with human α- thrombin at an enzyme to substrate weight ratio of 1:50 resulted in the formation of activated protein C, an enzyme with serine amidase activity. In the activation reaction, the apparent molecular weight of the heavy chain decreased from 41,000 to 40,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. No apparent change in the molecular weight of the light chain was observed in the activation process. The heavy chain of human activated protein C also contains the active-site serine residue as evidenced by its ability to react with radiolabeled diisopropyl fluorophosphate. Human activated protein C markedly […] Find the latest version: https://jci.me/109521/pdf Human Plasma Protein C ISOLATION, CHARACTERIZATION, AND MECHANISM OF ACTIVATION BY a-THROMBIN WALTER KISIEL, Department of Biochemistry, University of Washingtont, Seattle, Washington 98195 A B S TR A C T Protein C is a vitamin K-dependent like its bovine counterpart, exists in plasma as a zymo- protein, which exists in bovine plasma as a precursor gen and is converted to a serine protease by limited of a serine protease. In this study, protein C was iso- proteolysis with attendant anticoagulant activity. lated to homogeneity from human plasma by barium citrate adsorption and elution, ammonium sulfate frac- INTRODUCTION tionation, DEAE-Sephadex chromatography, dextran sulfate agarose chromatography, and preparative poly- Earlier work has demonstrated that protein C is a vita- acrylamide gel electrophoresis. Human protein C (M, min K-dependent glycoprotein, which exists in bovine = 62,000) contains 23% carbohydrate and is composed plasma as a precursor of a serine protease (2-4). Bovine of a light chain (M, = 21,000) and a heavy chain (Mr protein C is composed of a heavy chain (Mr = 41,000) = 41,000) held together by a disulfide bond(s). The and a light chain (Mr = 21,000) held together by a di- light chain has an amino-terminal sequence of Ala- sulfide bond(s). Incubation of bovine protein C vith Asn-Ser-Phe-Leu- and the heavy chain has an amino- either a-thrombin or a protease from Russell's viper terminal sequence of Asp-Pro-Glu-Asp-Gln. The resi- venom (RVV-X)l resulted in the cleavage of an Arg-Ile dues that are identical to bovine protein C are under- peptide bond between residues 14 and 15 of the heavy lined. Incubation of human protein C with human chain with the concomitant activation of the zymogen. a-thrombin at an enzyme to substrate weight ratio of The heavy chain of activated protein C contains the 1:50 resulted in the formation of activated protein C, amino-terminal sequence of Ile-Val-Asp-Gly and an an enzyme with serine amidase activity. In the activa- active-site sequence homologous with a number of tion reaction, the apparent molecular weight of the plasma serine proteases (4). heavy chain decreased from 41,000 to 40,000 as de- Previous work from our laboratory has shown that termined by gel electrophoresis in the presence of bovine activated protein C exhibits anticoagulant ac- sodium dodecyl sulfate. No apparent change in the tivity in the presence of phospholipid and calcium ions molecular weight of the light chain was observed in the (5). From our present information, it appears highly activation process. The heavy chain of human activated probable that activated protein C is responsible for the protein C also contains the active-site serine residue anticoagulant activity described by Marciniak nearly as evidenced by its ability to react with radiolabeled a decade ago (6). Experiments employing homogeneous diisopropyl fluorophosphate. Human activated protein protein preparations indicate that the anticoagulant C markedly prolongs the kaolin-cephalin clotting time effect of activated protein C is caused in part by the of human plasma, but not that of bovine plasma. The inactivation of factor Va.2 This inactivation reaction amidolytic and anticoagulant activities of human acti- involves the proteolytic cleavage of specific polypep- vated protein C were completely obviated by prior in- tide chains in the factor Va molecule (8). The inactiva- cubation of the enzyme with diisopropyl fluorophos- tion of factor Va by activated protein C requires phos- phate. These results indicate that human protein C, pholipid and calcium ions, and was inhibited by prior I Abbreviations used in this paper: DFP, diisopropyl fluoro- A preliminary report of this work has appeared elsewhere (1). phosphate; Mes, 2-(N-morpholino)ethanesulfonic acid; RW-X, Dr. Kisiel is an Established Investigator of the American the protease from Russell's viper venom that activates factor X; Heart Association. SDS, sodium dodecyl sulfate. Received for publication 2 April 1979 and in revised form 2 The nomenclature for the various clotting factors is that 14 May 1979. recommended by an international nomenclature committee (7). J. Clin. Invest. © The American Society for Clinical Investigation, Inc. * 0021-973817910910761/09 $1.00 761 Volume 64 September 1979 761 -769 incubation of the enzyme with diisopropyl fluorophos- tein content was determined by amino acid analyses after phate (DFP) (8). hydrolyzing an aliquot of the sample in 6 N HCI for 24 h at protein 110°C in evacuated tubes. In this article, a procedure for the isolation of SDS-polyacrylamide gel electrophoresis was performed as C from human plasma is presented along with some of described (4) employing 10% polyacrylamide gels. the molecular properties of the purified protein. In Amino acid analyses were carried out by standard proce- addition, the mechanism of activation of human protein dures (18, 19) employing a Durrum model D500 amino acid C by thrombin is described. This mechanism is essen- analyzer (Durrum Instrument Corp., Sunnyvale, Calif.). Tryp- tophan was determined by the procedure of Hugli and Moore tially identical with that previously observed for bovine (20), and half-cystine was determined as cysteic acid according protein C when it is activated by either trypsin (3, 4), to Hirs (21). RW-X (4), or a-thrombin (5). Neuraminic acid was determined according to Warren (22). Hexose and hexosamine were determined after hydrolysis of METHODS the sample (500-1,000 ,ug) in 90% glacial acetic acid contain- ing 0.5 N H2SO4 for 8 h at 80°C under a nitrogen atmosphere. DEAE-Sephadex A-50, SP-Sephadex C-50, Sephadex G-150, Inositol was added to the sample to serve as an internal stand- Sephadex G-50, Sephadex G-15, Sepharose 4B and dextran ard. After hydrolysis, the sugars were reduced and acetylated sulfate were products of Pharmacia Fine Chemicals, Piscataway, according to Yang and Hakomori (23). Sugars were identified N. J. Imidazole (Grade I), soybean trypsin inhibitor (type and quantitated by gas chromatography and mass spectrometry II-S), bovine serum albumin, ovalbumin, carbonic anhydrase, employing a gas chromatograph (model 402; Hewlett-Packard myoglobin, Coomassie Brilliant Blue R, dithiothreitol, lyo- Co., Palo Alto, Calif.) and a gas chromatograph-mass spec- philized Vipera russelli venom, thiobarbituric acid, N-acetyl- trometer (model 330; Finnigan Corp., Sunnyvale, Calif.). neuraminic acid, factor II-deficient bovine plasma, factor Glass columns (6 ft x Ys in) containing 3% OV-225 on Supel- X-deficient bovine plasma, and Tris (Trizma) base were ob- coport were used (Supelco Inc., Bellefonte, Pa.). The columns tained from Sigma Chemical Co., St. Louis, Mo. Kaolin (acid were usually programmed for 10 min at 1600C followed by a washed) was purchased from Fisher Scientific Co., Fair Lawn, 1°C/min rise to 210°C. N. J. Factor IX- and factor VII-deficient human plasmas were Automated Edman degradations were performed with a obtained from George King Bio-Medical, Inc., Overland Park, Beckman sequencer (model 890C; Beckman Instruments, Kans. Benzamidine hydrochloride, DFP, cyclohexanone, and Inc., Fullerton, Calif.) by a modification of the technique de- 4-vinyl pyridine were purchased from Aldrich Chemical Co., scribed by Edman and Begg (24). Dilute Quadrol (0.1 M; Milwaukee, Wis. Ammonium sulfate (enzyme grade) and BASF Wyandotte Corp., Industrial Chemicals Group; Wyan- guanidine hydrochloride (ultrapure) were supplied by dotte, Mich.) was employed as the coupling buffer. Phenyl- Schwarz/Mann Div. Becton, Dickinson & Co., Orangeburg, thiohydantoin (Pth) amino acids were identified and quanti- N. Y., Sodium dodecyl sulfate (SDS) was obtained from British tated by high pressure liquid chromatography (25, 26). For Drug House, Poole, England. Acrylamide, N,N'-methylene- amino-terminal analyses, -3 mg of the S-pyridylethylated bisacrylamide, N,N,N',N'-tetramethylethylenediamine, and heavy chain of protein C and 2 mg of the S-pyridylethylated urea (electrophoresis grade) were purchased from Bio-Rad light chain of protein C were used. All analyses were per- Laboratories, Richmond, Calif. Diisopropyl N-[1-3H]fluoro- formed in duplicate. phosphate (0.9 Ci/mmol) was obtained from New England Amidase activity of activated protein C was measured as Nuclear, Boston, Mass.
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