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. 1994 Apr 12;91(8):3353–3357. doi: 10.1073/pnas.91.8.3353

Molecular cloning, expression, and partial characterization of a second human tissue-factor-pathway inhibitor.

C A Sprecher 1, W Kisiel 1, S Mathewes 1, D C Foster 1
PMCID: PMC43575  PMID: 8159751

Abstract

Previous studies have shown that tissue-factor-pathway inhibitor (TFPI) is an important regulator of the extrinsic pathway of blood coagulation through its ability to inhibit factor Xa and factor VIIa-tissue factor activity. We describe the molecular cloning and expression of a full-length cDNA that encodes a molecule, designated TFPI-2, that has a similar overall domain organization and considerable primary amino acid sequence homology to TFPI. After a 22-residue signal peptide, the mature protein contains 213 amino acids with 18 cysteines and two canonical N-linked glycosylation sites. The deduced sequence of mature TFPI-2 revealed a short acidic amino-terminal region, three tandem Kunitz-type domains, and a carboxyl-terminal tail highly enriched in basic amino acids. Northern analysis indicates that TFPI-2 is transcribed in umbilical vein endothelial cells, liver, and placenta. TFPI-2 was expressed in baby hamster kidney cells and purified from the serum-free conditioned medium by a combination of heparin-agarose chromatography, Mono Q FPLC, Mono S FPLC, and Superose 12 FPLC. Purified TFPI-2 migrated as a single band in SDS/PAGE and exhibited a molecular mass of 32 kDa in the presence and absence of reducing agent. The amino-terminal sequence of recombinant TFPI-2 was identical to that predicted from the cDNA. Despite its structural similarity to TFPI, the purified recombinant TFPI-2 failed to react with polyclonal anti-TFPI IgG. Preliminary studies indicated that purified recombinant TFPI-2 strongly inhibited the amidolytic activities of trypsin and the factor VIIa-tissue factor complex. In addition, the inhibition of factor VIIa-tissue factor amidolytic activity by recombinant TFPI-2 was markedly enhanced in the presence of heparin. TFPI-2 at high concentrations weakly inhibited the amidolytic activity of human factor Xa, but had no measurable effect on the amidolytic activity of human thrombin.

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Selected References

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  1. Bajaj M. S., Kuppuswamy M. N., Saito H., Spitzer S. G., Bajaj S. P. Cultured normal human hepatocytes do not synthesize lipoprotein-associated coagulation inhibitor: evidence that endothelium is the principal site of its synthesis. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8869–8873. doi: 10.1073/pnas.87.22.8869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Broze G. J., Jr The role of tissue factor pathway inhibitor in a revised coagulation cascade. Semin Hematol. 1992 Jul;29(3):159–169. [PubMed] [Google Scholar]
  3. Callander N. S., Rao L. V., Nordfang O., Sandset P. M., Warn-Cramer B., Rapaport S. I. Mechanisms of binding of recombinant extrinsic pathway inhibitor (rEPI) to cultured cell surfaces. Evidence that rEPI can bind to and inhibit factor VIIa-tissue factor complexes in the absence of factor Xa. J Biol Chem. 1992 Jan 15;267(2):876–882. [PubMed] [Google Scholar]
  4. Foster D. C., Holly R. D., Sprecher C. A., Walker K. M., Kumar A. A. Endoproteolytic processing of the human protein C precursor by the yeast Kex2 endopeptidase coexpressed in mammalian cells. Biochemistry. 1991 Jan 15;30(2):367–372. doi: 10.1021/bi00216a009. [DOI] [PubMed] [Google Scholar]
  5. Girard T. J., Warren L. A., Novotny W. F., Likert K. M., Brown S. G., Miletich J. P., Broze G. J., Jr Functional significance of the Kunitz-type inhibitory domains of lipoprotein-associated coagulation inhibitor. Nature. 1989 Apr 6;338(6215):518–520. doi: 10.1038/338518a0. [DOI] [PubMed] [Google Scholar]
  6. Hamamoto T., Yamamoto M., Nordfang O., Petersen J. G., Foster D. C., Kisiel W. Inhibitory properties of full-length and truncated recombinant tissue factor pathway inhibitor (TFPI). Evidence that the third Kunitz-type domain of TFPI is not essential for the inhibition of factor VIIa-tissue factor complexes on cell surfaces. J Biol Chem. 1993 Apr 25;268(12):8704–8710. [PubMed] [Google Scholar]
  7. Kam C. M., Vlasuk G. P., Smith D. E., Arcuri K. E., Powers J. C. Thioester chromogenic substrates for human factor VIIa: substituted isocoumarins are inhibitors of factor VIIa and in vitro anticoagulants. Thromb Haemost. 1990 Aug 13;64(1):133–137. [PubMed] [Google Scholar]
  8. Kondo S., Kisiel W. Evidence that plasma lipoproteins inhibit the factor VIIa-tissue factor complex by a different mechanism that extrinsic pathway inhibitor. Blood. 1987 Dec;70(6):1947–1954. [PubMed] [Google Scholar]
  9. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  10. Laros R. K., Jr, Alger L. S. Thromboembolism and pregnancy. Clin Obstet Gynecol. 1979 Dec;22(4):871–878. doi: 10.1097/00003081-197912000-00008. [DOI] [PubMed] [Google Scholar]
  11. Nordfang O., Bjørn S. E., Valentin S., Nielsen L. S., Wildgoose P., Beck T. C., Hedner U. The C-terminus of tissue factor pathway inhibitor is essential to its anticoagulant activity. Biochemistry. 1991 Oct 29;30(43):10371–10376. doi: 10.1021/bi00107a002. [DOI] [PubMed] [Google Scholar]
  12. Paborsky L. R., Tate K. M., Harris R. J., Yansura D. G., Band L., McCray G., Gorman C. M., O'Brien D. P., Chang J. Y., Swartz J. R. Purification of recombinant human tissue factor. Biochemistry. 1989 Oct 3;28(20):8072–8077. doi: 10.1021/bi00446a016. [DOI] [PubMed] [Google Scholar]
  13. Pedersen A. H., Lund-Hansen T., Bisgaard-Frantzen H., Olsen F., Petersen L. C. Autoactivation of human recombinant coagulation factor VII. Biochemistry. 1989 Nov 28;28(24):9331–9336. doi: 10.1021/bi00450a013. [DOI] [PubMed] [Google Scholar]
  14. Pedersen A. H., Nordfang O., Norris F., Wiberg F. C., Christensen P. M., Moeller K. B., Meidahl-Pedersen J., Beck T. C., Norris K., Hedner U. Recombinant human extrinsic pathway inhibitor. Production, isolation, and characterization of its inhibitory activity on tissue factor-initiated coagulation reactions. J Biol Chem. 1990 Oct 5;265(28):16786–16793. [PubMed] [Google Scholar]
  15. Rapaport S. I. Inhibition of factor VIIa/tissue factor-induced blood coagulation: with particular emphasis upon a factor Xa-dependent inhibitory mechanism. Blood. 1989 Feb;73(2):359–365. [PubMed] [Google Scholar]
  16. Sandset P. M., Warn-Cramer B. J., Rao L. V., Maki S. L., Rapaport S. I. Depletion of extrinsic pathway inhibitor (EPI) sensitizes rabbits to disseminated intravascular coagulation induced with tissue factor: evidence supporting a physiologic role for EPI as a natural anticoagulant. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):708–712. doi: 10.1073/pnas.88.3.708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Sprecher C. A., Grant F. J., Grimm G., O'Hara P. J., Norris F., Norris K., Foster D. C. Molecular cloning of the cDNA for a human amyloid precursor protein homolog: evidence for a multigene family. Biochemistry. 1993 May 4;32(17):4481–4486. doi: 10.1021/bi00068a002. [DOI] [PubMed] [Google Scholar]
  19. Wesselschmidt R., Likert K., Girard T., Wun T. C., Broze G. J., Jr Tissue factor pathway inhibitor: the carboxy-terminus is required for optimal inhibition of factor Xa. Blood. 1992 Apr 15;79(8):2004–2010. [PubMed] [Google Scholar]
  20. Wun T. C., Kretzmer K. K., Girard T. J., Miletich J. P., Broze G. J., Jr Cloning and characterization of a cDNA coding for the lipoprotein-associated coagulation inhibitor shows that it consists of three tandem Kunitz-type inhibitory domains. J Biol Chem. 1988 May 5;263(13):6001–6004. [PubMed] [Google Scholar]
  21. von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 1986 Jun 11;14(11):4683–4690. doi: 10.1093/nar/14.11.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]

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