Investigating clearance mechanisms for recombinant activated factor VII in a perfused liver model

 

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Summary

Clearance mechanisms for recombinant activated human FVII (rFVIIa; NovoSeven®), a heterogeneously glycosylated protein, have yet to be fully elucidated, but may involve the liver. The effects of the γ-carboxy glutamic acid (Gla) domain and the sialic acid content of the protein on rFVIIa clearance were investigated following intravenous administration of rFVIIa lacking the Gla domain, des(1–44) rFVIIa and asialo-rFVIIa in pharmacokinetic (PK) studies and perfused rat livers. PK parameters for both rFVIIa and des(1–44) rFVIIa had similar biphasic clearance profiles, as well as half-lives ([t½]=80 and 88 minutes, respectively), while asialo-rFVIIa was cleared quickly (t½=21 minutes) with a linear clearance profile. Perfused liver studies with all proteins (10 nM) mirrored the trends in profiles observed in the PK study. rFVIIa and des(1–44) rFVIIa were cleared to a similar extent, 41% and 35%, respectively, after 1 h, whereas plasma-derived FVII from humans (which has a higher sialylation content than rFVIIa) was cleared to a lesser extent (21%). Asialo-rFVIIa, on the other hand, was almost totally cleared and when an excess of asialo-orosomucoid was added to the perfusate, its clearance was significantly reduced (by 34%) and also for rFVIIa, albeit to a lesser extent (by 14%). Together these data suggest that carbohydrate receptor(s) (e.g. the asialoglycoprotein receptor, ASGPR) play a role in asialo-rFVIIa and rFVIIa clearance. In vivo and liver clearance data correlated well showing similar trends and indi-cated that rFVIIa clearance is not affected by the Gla domain, but rather by a subpopulation of N-glycosylated structures on rFVIIa.

• References

• 1 Neuenschwander PF, Morrissey JH. Roles of the membrane-interactive regions of factor VIIa and tissue factor. The factor VIIa Gla domain is dispensable for binding to tissue factor but important for activation of factor X. J Biol Chem 1994; 269: 8007-8013.
  MissingFormLabel

  PubMedSearch in Google Scholar
• 2 Tranholm M, Kristensen K, Kristensen AT. et al. Improved hemostasis with super-active analogs of factor VIIa in a mouse model of hemophilia A. Blood 2003; 102: 3615-3620.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 3 Fuchs HE, Trapp HG, Griffith MJ. et al. Regulation of factor IXa in vitro in human and mouse plasma and in vivo in the mouse. Role of the endothelium and the plasma proteinase inhibitors. J Clin Invest 1984; 73: 1696-1703.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 4 Narita M, Rudolph AE, Miletich JP. et al. The low-density lipoprotein receptor-related protein (LRP) mediates clearance of coagulation factor Xa in vivo. Blood 1998; 91: 555-560.
  MissingFormLabel

  PubMedSearch in Google Scholar
• 5 Chang GT, Kisiel W. Internalization and degradation of recombinant human coagulation factor VIIa by the human hepatoma cell line HuH7. Thromb Haemost 1995; 73: 231-238.
  MissingFormLabel

  Thieme ConnectPubMedSearch in Google Scholar
• 6 Hjortoe G, Sorensen BB, Petersen LC. et al. Factor VIIa binding and internalization in hepatocytes. Thromb Haemost 2005; 03: 2264-2273.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 7 Rao LV, Pendurthi UR. Regulation of tissue factor-factor VIIa expression on cell surfaces: a role for tissue factor-factor VIIa endocytosis. Mol Cell Biochem 2003; 253: 131-140.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 8 Rao LV, Pendurthi UR. Factor VIIa binding to endothelial cell protein C receptor. Thromb Res 2008; 122 (Suppl. 01) S3-S6.
  MissingFormLabel

  PubMedSearch in Google Scholar
• 9 Weeterings C, de Groot PG, Adelmeijer J. et al. The glycoprotein Ib-IX-V complex contributes to tissue factor-independent thrombin generation by recombinant factor VIIa on the activated platelet surface. Blood 2008; 112: 3227-3233.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 10 Beeby TL, Chasseaud LF, Taylor T. et al. Distribution of the recombinant coagulation factor 125I-rFVIIa in rats. Thromb Haemost 1993; 70: 465-468.
  MissingFormLabel

  Thieme ConnectPubMedSearch in Google Scholar
• 11 Thomsen MK, Diness V, Nilsson P. et al. Pharmacokinetics of recombinant factor VIIa in the rat--a comparison of bio-, immuno- and isotope assays. Thromb Hae-most 1993; 70: 458-464.
  MissingFormLabel

  Thieme ConnectPubMedSearch in Google Scholar
• 12 Seested T, Nielsen HM, Christensen EI. et al. Cellular and intracellular distribution of recombinant activated factor VII in the rat liver. Thromb Haemost 2010; 103: 860-862.
  MissingFormLabel

  Thieme ConnectPubMedSearch in Google Scholar
• 13 Skogh T, Blomhoff R, Eskild W, Berg T. Hepatic uptake of circulating IgG immune complexes. Immunology 1985; 55: 585-594.
  MissingFormLabel

  PubMedSearch in Google Scholar
• 14 Moestrup SK, Gliemann J, Pallesen G. Distribution of the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein in human tissues. Cell Tissue Res 1992; 269: 375-382.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 15 Smedsrod B, Pertoft H, Gustafson S. et al. Scavenger functions of the liver endothelial cell. Biochem J 1990; 266: 313-327.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 16 Appa R, Christensen MS. Investigation of human recombinant FVIIa metabolism in a perfused rat liver model. Thromb Haemost 2007; 05 (Suppl. 02) P-T-054. Abstract
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 17 Nicolaisen EM, Petersen LC, Thim L. et al. Generation of Gladomainless FVIIa by cathepsin G-mediated cleavage. FEBS Lett 1992; 306: 157-160.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 18 Eap CB, Baumann P. Isoelectric focusing of alpha-1 acid glycoprotein (orosomucoid) in immobilized pH-gradients with 8M urea: detection of its desialylated variants using an alkaline phosphatase-linked secondary antibody system. Electrophoresis 1988; 09: 650-654.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 19 Behrens C, Garibay PW, Zundel M. Use of galactose oxidase for selective chemical conjugation of protractor molecules to proteins of therapeutic interest. 2005. International Patent application number WO200501435.
  MissingFormLabel

  Search in Google Scholar
• 20 Meijer DK, Keulemans K, Mulder GJ. Isolated perfused rat liver technique. Meth Enzymol 1981; 77: 81-94.
  MissingFormLabel

  PubMedSearch in Google Scholar
• 21 Wolkoff AW, Johansen KL, Goeser T. The isolated perfused rat liver: preparation and application. Anal Biochem 1987; 167: 1-14.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 22 Wildgoose P, Nemerson Y, Hansen LL. et al. Measurement of basal levels of factor VIIa in hemophilia A and B patients. Blood 1992; 80: 25-28.
  MissingFormLabel

  PubMedSearch in Google Scholar
• 23 Morrissey JH, Macik BG, Neuenschwander PF. et al. Quantitation of activated factor VII levels in plasma using a tissue factor mutant selectively deficient in promoting factor VII activation. Blood 1993; 81: 734-744.
  MissingFormLabel

  PubMedSearch in Google Scholar
• 24 Klausen NK, Bayne S, Palm L. Analysis of the site-specific asparagine-linked glycosylation of recombinant human coagulation factor VIIa by glycosidase digestions, liquid chromatography, and mass spectrometry. Mol Biotechnol 1998; 09: 195-204.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 25 Klausen NK, Kornfelt T. Analysis of the glycoforms of human recombinant factor VIIa by capillary electrophoresis and high-performance liquid chromatography. J Chromatogr A 1995; 718: 195-202.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 26 Ashwell G, Harford J. Carbohydrate-specific receptors of the liver. Annu Rev Biochem 1982; 51: 531-554.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 27 Baenziger JU, Fiete D. Galactose and N-acetylgalactosamine-specific endocytosis of glycopeptides by isolated rat hepatocytes. Cell 1980; 22: 611-620.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 28 Schwartz AL. The hepatic asialoglycoprotein receptor. CRC Crit Rev Biochem 1984; 16: 207-233.
  MissingFormLabel

  PubMedSearch in Google Scholar
• 29 Schiff JM, Huling SL, Jones AL. Receptor-mediated uptake of asialoglycoprotein by the primate liver initiates both lysosomal and transcellular pathways. Hepatology 1986; 06: 837-847.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 30 Ashwell G, Morell AG. The role of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Adv Enzymol Relat Areas Mol Biol 1974; 41: 99-128.
  MissingFormLabel

  PubMedSearch in Google Scholar
• 31 Wall DA, Wilson G, Hubbard AL. The galactose-specific recognition system of mammalian liver: the route of ligand internalization in rat hepatocytes. Cell 1980; 21: 79-93.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 32 Wall DA, Hubbard AL. Receptor-mediated endocytosis of asialoglycoproteins by rat liver hepatocytes: biochemical characterization of the endosomal compartments. J Cell Biol 1985; 101: 2104-2112.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 33 Morell AG, Gregoriadis G, Scheinberg IH. et al. The role of sialic acid in determining the survival of glycoproteins in the circulation. J Biol Chem 1971; 246: 1461-1467.
  MissingFormLabel

  PubMedSearch in Google Scholar
• 34 Townsend RR, Wall DA, Hubbard AL. et al. Rapid release of galactose-terminated ligands after endocytosis by hepatic parenchymal cells: Evidence for a role of carbohydrate structure in the release of internalized ligand from receptor. PNAS 1984; 81: 466-470.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 35 Pricer Jr. WE, Ashwell G. The binding of desialylated glycoproteins by plasma membranes of rat liver. J Biol Chem 1971; 246: 4825-4833.
  MissingFormLabel

  PubMedSearch in Google Scholar
• 36 Jones AJ, Papac DI, Chin EH. et al. Selective clearance of glycoforms of a complex glycoprotein pharmaceutical caused by terminal N-acetylglucosamine is similar in humans and cynomolgus monkeys. Glycobiology 2007; 17: 529-540.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 37 Webster R, Taberner J, Edgington A. et al. Role of sialylation in determining the pharmacokinetics of neutrophil inhibitory factor (NIF) in the Fischer 344 rat. Xenobiotica 1999; 29: 1141-1155.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 38 Thim L, Bjoern S, Christensen M. et al. Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells. Biochemistry 1988; 27: 7785-7793.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 39 Jurlander B, Thim L, Klausen NK. et al. Recombinant activated factor VII (rFVIIa): Characterization, manufacturing, and clinical development. Semin Thromb Hemost 2001; 27: 373-383.
  MissingFormLabel

  Thieme ConnectPubMedSearch in Google Scholar
• 40 Weigel PH. Endocytosis and function of the hepatic asialoglycoprotein receptor. Subcell Biochem 1993; 19: 125-161.
  MissingFormLabel

  PubMedSearch in Google Scholar
• 41 Zeng FY, Oka JA, Weigel PH. A specific antibody to the carbohydrate recognition domain of the asialoglycoprotein receptor RHL1 subunit does not react with RHL2/3 but blocks ligand binding. Bichem Biophys Res Commun 1998; 249: 236-240.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 42 Townsend R, Stahl P. Isolation and characterization of a mannose/N-acetylgluco-samine/fucose-binding protein from rat liver. Biochem J 1981; 194: 209-214.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 43 Pricer Jr. WE, Hudgin RL, Ashwell G. et al. A membrane receptor protein for asialoglycoproteins. Meth Enzymol 1974; 34: 688-691.
  MissingFormLabel

  PubMedSearch in Google Scholar
• 44 Thomas P, Summer JW. The biliary excretion of circulating asialoglycoproteins in the rat. Biochem Biophys Res Commun 2009; 80: 335-339.
  MissingFormLabel

  PubMedSearch in Google Scholar
• 45 Schiff JM, Fisher MM, Underdown BJ. Receptor-mediated biliary transport of immunoglobulin A and asialoglycoprotein: Sorting and missorting of ligands revealed by two radiolabeling methods. J Cell Biol 1984; 98: 79-89.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 46 Thomas P, Toth CA, Zamcheck N. The mechanism of bilary excretion of alpha-1 acid glyco protein in the rat. Gastroenterology 1981; 80: 1302
  MissingFormLabel

  PubMedSearch in Google Scholar
• 47 Iakhiaev A, Pendurthi UR, Voigt J. et al. Catabolism of factor VIIa bound to tissue factor in fibroblasts in the presence and absence of tissue factor pathway inhibitor. J Biol Chem 1999; 274: 36995-37003.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 48 Ghosh S, Pendurthi UR, Steinoe A. et al. Endothelial cell protein C receptor acts as a cellular receptor for factor VIIa on endothelium. J Biol Chem 2007; 282: 11849-11857.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar
• 49 Fleck RA, Rao LV, Rapaport SI. et al. Localization of human tissue factor antigen by immunostaining with monospecific, polyclonal anti-human tissue factor anti-body. Thromb Res 1990; 59: 421-437.
  MissingFormLabel

  CrossrefPubMedSearch in Google Scholar