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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1984 Aug;81(15):4766–4770. doi: 10.1073/pnas.81.15.4766

Characterization of a cDNA coding for human protein C.

D Foster, E W Davie
PMCID: PMC391571  PMID: 6589623

Abstract

Protein C is a precursor to a serine protease that is present in mammalian plasma. In its activated form, it readily inactivates factor Va and factor VIIIa, two proteins that participate as cofactors in the blood coagulation cascade. In the present studies, a lambda gt11 library containing cDNA inserts prepared from human liver mRNA has been screened with an antibody to human protein C. Seven positive clones were isolated from 2 X 10(6) phage and were plaque-purified. The cDNA inserts of two of these phage were sequenced and shown to code for human protein C. Each cDNA insert coded for a portion of the light chain of the molecule, a connecting region, the heavy chain, a stop codon, a 3'-noncoding region, and a poly(A) tail. The length of the noncoding sequence on the 3' end differed in the two clones, but each contained a processing or polyadenylylation signal followed by a poly(A) tail. The amino acid sequence as determined from the cDNA indicates that protein C is synthesized as a single-chain polypeptide containing the light chain and the heavy chain connected by a dipeptide of Lys-Arg. The single-chain molecule is then converted to the light and heavy chains by cleavage of two or more internal peptide bonds. In plasma, the heavy and light chains of protein C are linked together by a disulfide bond. The amino acid sequence of human protein C shows a high degree of homology with that of the bovine molecule. The DNA sequence coding for the catalytic region near the active site serine in human protein C also showed a high degree of DNA and amino acid sequence identity with prothrombin, factor IX, and factor X, three of the other vitamin K-dependent serine proteases that are present in plasma.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Biggin M. D., Gibson T. J., Hong G. F. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. doi: 10.1073/pnas.80.13.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Canfield W. M., Kisiel W. Evidence of normal functional levels of activated protein C inhibitor in combined Factor V/VIII deficiency disease. J Clin Invest. 1982 Dec;70(6):1260–1272. doi: 10.1172/JCI110725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dahlbäck B., Stenflo J. Inhibitory effect of activated protein C on activation of prothrombin by platelet-bound factor Xa. Eur J Biochem. 1980 Jun;107(2):331–335. doi: 10.1111/j.1432-1033.1980.tb06033.x. [DOI] [PubMed] [Google Scholar]
  4. Degen S. J., MacGillivray R. T., Davie E. W. Characterization of the complementary deoxyribonucleic acid and gene coding for human prothrombin. Biochemistry. 1983 Apr 26;22(9):2087–2097. doi: 10.1021/bi00278a008. [DOI] [PubMed] [Google Scholar]
  5. DiScipio R. G., Davie E. W. Characterization of protein S, a gamma-carboxyglutamic acid containing protein from bovine and human plasma. Biochemistry. 1979 Mar 6;18(5):899–904. doi: 10.1021/bi00572a026. [DOI] [PubMed] [Google Scholar]
  6. Drakenberg T., Fernlund P., Roepstorff P., Stenflo J. beta-Hydroxyaspartic acid in vitamin K-dependent protein C. Proc Natl Acad Sci U S A. 1983 Apr;80(7):1802–1806. doi: 10.1073/pnas.80.7.1802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Esmon C. T., Owen W. G. Identification of an endothelial cell cofactor for thrombin-catalyzed activation of protein C. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2249–2252. doi: 10.1073/pnas.78.4.2249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Esmon C. T., Stenflo J., Suttie J. W. A new vitamin K-dependent protein. A phospholipid-binding zymogen of a serine esterase. J Biol Chem. 1976 May 25;251(10):3052–3056. [PubMed] [Google Scholar]
  9. Esmon N. L., Owen W. G., Esmon C. T. Isolation of a membrane-bound cofactor for thrombin-catalyzed activation of protein C. J Biol Chem. 1982 Jan 25;257(2):859–864. [PubMed] [Google Scholar]
  10. Fernlund P., Stenflo J. Amino acid sequence of the light chain of bovine protein C. J Biol Chem. 1982 Oct 25;257(20):12170–12179. [PubMed] [Google Scholar]
  11. Graves C. B., Munns T. W., Willingham A. K., Strauss A. W. Rat factor X is synthesized as a single chain precursor inducible by prothrombin fragments. J Biol Chem. 1982 Nov 10;257(21):13108–13113. [PubMed] [Google Scholar]
  12. Griffin J. H., Evatt B., Zimmerman T. S., Kleiss A. J., Wideman C. Deficiency of protein C in congenital thrombotic disease. J Clin Invest. 1981 Nov;68(5):1370–1373. doi: 10.1172/JCI110385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Griffin J. H., Mosher D. F., Zimmerman T. S., Kleiss A. J. Protein C, an antithrombotic protein, is reduced in hospitalized patients with intravascular coagulation. Blood. 1982 Jul;60(1):261–264. [PubMed] [Google Scholar]
  14. Kisiel W., Canfield W. M., Ericsson L. H., Davie E. W. Anticoagulant properties of bovine plasma protein C following activation by thrombin. Biochemistry. 1977 Dec 27;16(26):5824–5831. doi: 10.1021/bi00645a029. [DOI] [PubMed] [Google Scholar]
  15. Kisiel W., Ericsson L. H., Davie E. W. Proteolytic activation of protein C from bovine plasma. Biochemistry. 1976 Nov 2;15(22):4893–4900. doi: 10.1021/bi00667a022. [DOI] [PubMed] [Google Scholar]
  16. Kisiel W. Human plasma protein C: isolation, characterization, and mechanism of activation by alpha-thrombin. J Clin Invest. 1979 Sep;64(3):761–769. doi: 10.1172/JCI109521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kurachi K., Davie E. W. Isolation and characterization of a cDNA coding for human factor IX. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6461–6464. doi: 10.1073/pnas.79.21.6461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Leytus S. P., Chung D. W., Kisiel W., Kurachi K., Davie E. W. Characterization of a cDNA coding for human factor X. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3699–3702. doi: 10.1073/pnas.81.12.3699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Marlar R. A., Kleiss A. J., Griffin J. H. Mechanism of action of human activated protein C, a thrombin-dependent anticoagulant enzyme. Blood. 1982 May;59(5):1067–1072. [PubMed] [Google Scholar]
  20. McMullen B. A., Fujikawa K., Kisiel W. The occurrence of beta-hydroxyaspartic acid in the vitamin K-dependent blood coagulation zymogens. Biochem Biophys Res Commun. 1983 Aug 30;115(1):8–14. doi: 10.1016/0006-291x(83)90961-0. [DOI] [PubMed] [Google Scholar]
  21. Owen W. G., Esmon C. T. Functional properties of an endothelial cell cofactor for thrombin-catalyzed activation of protein C. J Biol Chem. 1981 Jun 10;256(11):5532–5535. [PubMed] [Google Scholar]
  22. Proudfoot N. J., Brownlee G. G. 3' non-coding region sequences in eukaryotic messenger RNA. Nature. 1976 Sep 16;263(5574):211–214. doi: 10.1038/263211a0. [DOI] [PubMed] [Google Scholar]
  23. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Seligsohn U., Berger A., Abend M., Rubin L., Attias D., Zivelin A., Rapaport S. I. Homozygous protein C deficiency manifested by massive venous thrombosis in the newborn. N Engl J Med. 1984 Mar 1;310(9):559–562. doi: 10.1056/NEJM198403013100904. [DOI] [PubMed] [Google Scholar]
  25. Staden R. Further procedures for sequence analysis by computer. Nucleic Acids Res. 1978 Mar;5(3):1013–1016. doi: 10.1093/nar/5.3.1013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Staden R. Sequence data handling by computer. Nucleic Acids Res. 1977 Nov;4(11):4037–4051. doi: 10.1093/nar/4.11.4037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Stenflo J. A new vitamin K-dependent protein. Purification from bovine plasma and preliminary characterization. J Biol Chem. 1976 Jan 25;251(2):355–363. [PubMed] [Google Scholar]
  28. Stenflo J., Fernlund P. Amino acid sequence of the heavy chain of bovine protein C. J Biol Chem. 1982 Oct 25;257(20):12180–12190. [PubMed] [Google Scholar]
  29. Vehar G. A., Davie E. W. Preparation and properties of bovine factor VIII (antihemophilic factor). Biochemistry. 1980 Feb 5;19(3):401–410. doi: 10.1021/bi00544a001. [DOI] [PubMed] [Google Scholar]
  30. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  31. Young R. A., Davis R. W. Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1194–1198. doi: 10.1073/pnas.80.5.1194. [DOI] [PMC free article] [PubMed] [Google Scholar]

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