Abstract
Coagulation is the complex process by which activation of plasmatic haemostasis proteins ends up with the generation of fibrin. Most of the plasma coagulation proteins are synthesized in hepatocytes. The aim of this chapter is to describe experimental procedures allowing to measure the secretion by primary human hepatocytes and functional activity (including production of fibrillar material from extracellular medium) of haemostasis proteins including factors II, V, VII, VIII, PIVKA-II (protein induced by vitK 1 absence or antagonist II), antithrombin and protein S. In addition, we show how treatments of hepatocyte cultures with vitamin K and/or warfarin affect the secretion of haemostasis proteins. The results demonstrate that primary cultures of human hepatocytes constitute an invaluable model for investigating haemostasis protein expression and activity and therapeutic strategies targeting these proteins.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Tripodi, A. and Mannucci, P.M. (2007) Abnormalities of hemostasis in chronic liver disease: reappraisal of their clinical significance and need for clinical and laboratory research. J. Hepatol. 46, 727–733.
Merli, G.J. and Fink, J. (2008) Vitamin K and thrombosis. Vitam. Horm. 78, 265–279.
Wallin, R., Wajih, N., and Hutson, S.M. (2008) VKORC1: a warfarin-sensitive enzyme in vitamin K metabolism and biosynthesis of vitamin K-dependent blood coagulation factors. Vitam. Horm. 78, 227–246.
D’Andrea, G., D’Ambrosio, R., and Margaglione, M. (2008) Oral anticoagulants: pharmacogenetics relationship between genetic and non-genetic factors. Blood Rev. 22, 127–140.
Schulman, S. and Bijsterveld, N.R. (2007) Anticoagulants and their reversal. Transfus. Med. Rev. 21, 37–48.
Heinrich, J. and George, J. (2001) Hemostatic abnormalities in liver and renal disease, in Hemostasis and Thrombosis (Colman, R.W., Hirsh, J., Marder, A.W., Clowes, A.W., and George, J.N., eds.), Lippincott Williams & Wilkins, Philadelphia, pp. 955–960.
Dodds, W.J. (1969) Storage, release, and synthesis of coagulation factors in isolated perfused organs. Am. J. Physiol. 217, 879–883.
Olson, J.P., Miller, L.L., and Troup, S.B. (1966) Synthesis of clotting factors by the isolated perfused rat liver. J. Clin. Invest. 45, 690–701.
Owen, C.A., Jr. and Bowie, E.J. (1977) Generation of coagulation factors V, XI, and XII by the isolated rat liver. Haemostasis 6, 205–212.
Gordon, E.M., Gallagher, C.A., Johnson, T.R., Blossey, B.K., and Ilan, J. (1990) Hepatocytes express blood coagulation factor XII (Hageman factor). J. Lab. Clin. Med. 115, 463–469.
Hoffman, M., Fuchs, H.E., and Pizzo, S.V. (1986) The macrophage-mediated regulation of hepatocyte synthesis of antithrombin III and alpha 1-proteinase inhibitor. Thromb. Res. 41, 707–715.
Mazzorana, M., Cornillon, B., Baffet, G., Hubert, N., Belleville, J., Eloy, R., and Guguen-Guillouzo, C. (1989) Biosynthesis of factor V by normal adult rat hepatocytes. Thromb. Res. 54, 655–675.
Odenthal, M., Neubauer, K., Baralle, F.E., Peters, H., Meyer zum Buschenfelde, K.H., and Ramadori, G. (1992) Rat hepatocytes in primary culture synthesize and secrete cellular fibronectin. Exp. Cell Res. 203, 289–296.
Otto, J.M., Grenett, H.E., and Fuller, G.M. (1987) The coordinated regulation of fibrinogen gene transcription by hepatocyte-stimulating factor and dexamethasone. J. Cell Biol. 105, 1067–1072.
Stamatoglou, S.C., Hughes, R.C., and Lindahl, U. (1987) Rat hepatocytes in serum-free primary culture elaborate an extensive extracellular matrix containing fibrin and fibronectin. J. Cell Biol. 105, 2417–2425.
Uno, S., Nakamura, M., Seki, T., and Ariga, T. (1997) Induction of tissue-type plasminogen activator (tPA) and type-1 plasminogen activator inhibitor (PAI-1) as early growth responses in rat hepatocytes in primary culture. Biochem. Biophys. Res. Commun. 239, 123–128.
Yagi, K., Yamada, C., Serada, M., Sumiyoshi, N., Michibayashi, N., Miura, Y., and Mizoguchi, T. (1995) Reciprocal regulation of prothrombin secretion and tyrosine aminotransferase induction in hepatocytes. Eur. J. Biochem. 227, 753–756.
Biron-Andreani, C., Bezat-Bouchahda, C., Raulet, E., Pichard-Garcia, L., Fabre, J.M., Saric, J., Baulieux, J., Schved, J.F., and Maurel, P. (2004) Secretion of functional plasma haemostasis proteins in long-term primary cultures of human hepatocytes. Br. J. Haematol. 125, 638–646.
Ferrini, J.B., Pichard, L., Domergue, J., and Maurel, P. (1997) Long-term primary cultures of adult human hepatocytes. Chem. Biol. Interact. 107, 31–45.
Ingerslev, J., Christiansen, B.S., Heickendorff, L., and Munck Petersen, C. (1988) Synthesis of factor VIII in human hepatocytes in culture. Thromb. Haemost. 60, 387–391.
Mazzorana, M., Baffet, G., Kneip, B., Launois, B., and Guguen-Guillouzo, C. (1991) Expression of coagulation factor V gene by normal adult human hepatocytes in primary culture. Br. J. Haematol. 78, 229–235.
Pichard, L., Raulet, E., Fabre, G., Ferrini, J.B., Ourlin, J.C., and Maurel, P. (2006) Human hepatocyte culture. Methods Mol. Biol. 320, 283–293.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Biron-Andréani, C., Raulet, E., Pichard-Garcia, L., Maurel, P. (2010). Use of Human Hepatocytes to Investigate Blood Coagulation Factor. In: Maurel, P. (eds) Hepatocytes. Methods in Molecular Biology, vol 640. Humana Press. https://doi.org/10.1007/978-1-60761-688-7_23
Download citation
DOI: https://doi.org/10.1007/978-1-60761-688-7_23
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60761-687-0
Online ISBN: 978-1-60761-688-7
eBook Packages: Springer Protocols