Download PDF
Thromb Haemost 2002; 88(03): 450-458
DOI: 10.1055/s-0037-1613237
Review Article
Schattauer GmbH

Expression and Characterization of Recombinant Murine Factor VIII

Christopher B. Doering
1   Winship Cancer Institute, Emory University, Atlanta, Georgia, USA
,
Ernest T. Parker
1   Winship Cancer Institute, Emory University, Atlanta, Georgia, USA
,
John F. Healey
1   Winship Cancer Institute, Emory University, Atlanta, Georgia, USA
,
Heather N. Craddock
1   Winship Cancer Institute, Emory University, Atlanta, Georgia, USA
,
Rachel T. Barrow
1   Winship Cancer Institute, Emory University, Atlanta, Georgia, USA
,
Pete Lollar
1   Winship Cancer Institute, Emory University, Atlanta, Georgia, USA
› Author Affiliations
Further Information

Publication History

Received 06 December 2001

Accepted after revision 20 May 2002

Publication Date:
08 December 2017 (online)

Also available at
    Permissions and Reprints

Summary

Hemophilia A is the inherited bleeding disorder that results from mutation of blood coagulation factor VIII (fVIII). Described here is the generation of a regulated expression system producing recombinant murine fVIII. Murine B-domainless fVIII was expressed at a peak level of 4 units/106 cells/24 h in serum-free media. Subsequently, a two-step purification procedure resulted in 5,300-fold enrichment and a 70% yield. Highly purified recombinant murine fVIII had a specific coagulant activity of 660 units per nanomole. It underwent proteolytic processing by thrombin to yield an activated heterotrimer that demonstrated significantly greater stability than activated human fVIII. Recombinant murine fVIII was utilized to generate an anti-fVIII polyclonal antibody. Intravenous injection of recombinant murine fVIII into hemophilia A mice failed to induce a significant anti-fVIII immune response using a schedule that yielded high titer inhibitory antibodies to human fVIII. This may provide an important model for the study of immune tolerance to fVIII.

Keywords

Recombinant - factor VIII - mouse - doxycycline-controlled expression
 

  • References

  • 1 Hoeben RC, Fallaux FJ, Schagen FH, van der Eb MM, van Ormondt H, van der EA. Factors impeding efficient expression of factor VIII complementary DNA minigenes. Blood Coagul Fibrinolysis 1997; 08 (Suppl. 02) S15-21.
    PubMedGoogle Scholar
  • 2 Plantier JL, Rodriguez MH, Enjolras N, Attali O, Negrier C. A factor VIII minigene comprising the truncated intron I of factor IX highly improves the in vitro production of factor VIII. Thromb Haemost 2001; 86 (02) 596-603.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 3 Chuah MK, Vanden TDriessche, Morgan RA. Development and analysis of retroviral vectors expressing human factor VIII as a potential gene therapy for hemophilia A. Hum Gene Ther 1995; 06 (11) 1363-77.
    CrossrefPubMedGoogle Scholar
  • 4 Lynch CM, Israel DI, Kaufman RJ, Miller AD. Sequences in the coding region of clotting factor VIII act as dominant inhibitors of RNA accumulation and protein production. Hum Gene Ther 1993; 04 (03) 259-72.
    CrossrefPubMedGoogle Scholar
  • 5 Bi L, Lawler AM, Antonarakis SE, High KA, Gearhart JD, Kazazian Jr HH. Targeted disruption of the mouse factor VIII gene produces a model of haemophilia A. Nat Genet 1995; 10 (01) 119-21.
    CrossrefPubMedGoogle Scholar
  • 6 Bi L, Sarkar R, Naas T, Lawler AM, Pain J, Shumaker SL, Bedian V, Kazazian Jr HH. Further characterization of factor VIII-deficient mice created by gene targeting: RNA and protein studies. Blood 1996; 88 (09) 3446-50.
    PubMedGoogle Scholar
  • 7 Balague C, Zhou J, Dai Y, Alemany R, Josephs SF, Andreason G, Hariharan M, Sethi E, Prokopenko E, Jan HY, Lou YC, Hubert-Leslie D, Ruiz L, Zhang WW. Sustained high-level expression of full-length human factor VIII and restoration of clotting activity in hemophilic mice using a minimal adenovirus vector. Blood 2000; 95 (03) 820-8.
    PubMedGoogle Scholar
  • 8 Bristol JA, Shirley P, Idamakanti N, Kaleko M, Connelly S. In vivo dose threshold effect of adenovirus-mediated factor VIII gene therapy in hemophiliac mice. Mol Ther 2000; 02 (03) 223-32.
    CrossrefPubMedGoogle Scholar
  • 9 Bristol JA, Gallo-Penn A, Andrews J, Idamakanti N, Kaleko M, Connelly S. Adenovirus-mediated factor VIII gene expression results in attenuated anti-factor VIII-specific immunity in hemophilia a mice compared with factor VIII protein infusion. Hum Gene Ther 2001; 12 (13) 1651-61.
    CrossrefPubMedGoogle Scholar
  • 10 Connelly S, Andrews JL, Gallo AM, Kayda DB, Qian J, Hoyer L, Kadan MJ, Gorziglia MI, Trapnell BC, McClelland A, Kaleko M. Sustained phenotypic correction of murine hemophilia A by in vivo gene therapy. Blood 1998; 91 (09) 3273-81.
    PubMedGoogle Scholar
  • 11 Lipshutz GS, Sarkar R, Flebbe-Rehwaldt L, Kazazian H, Gaensler KM. Short-term correction of factor VIII deficiency in a murine model of hemophilia A after delivery of adenovirus murine factor VIII in utero. Proc Natl Acad Sci USA 1999; 96 (23) 13324-9.
    CrossrefPubMedGoogle Scholar
  • 12 Sarkar R, Gao GP, Chirmule N, Tazelaar J, Kazazian Jr HH. Partial correction of murine hemophilia A with neo-antigenic murine factor VIII. Hum Gene Ther 2000; 11 (06) 881-94.
    CrossrefPubMedGoogle Scholar
  • 13 Vanden TDriessche, Vanslembrouck V, Goovaerts I, Zwinnen H, Vanderhaeghen ML, Collen D, Chuah MK. Long-term expression of human coagulation factor VIII and correction of hemophilia A after in vivo retroviral gene transfer in factor VIII-deficient mice. Proc Natl Acad Sci USA 1999; 96 (18) 10379-84.
    CrossrefPubMedGoogle Scholar
  • 14 Qian J, Borovok M, Bi L, Kazazian Jr HH, Hoyer LW. Inhibitor antibody development and T cell response to human factor VIII in murine hemophilia A. Thromb Haemost 1999; 81 (02) 240-4.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 15 Qian J, Collins M, Sharpe AH, Hoyer LW. Prevention and treatment of factor VIII inhibitors in murine hemophilia A. Blood 2000; 95 (04) 1324-9.
    PubMedGoogle Scholar
  • 16 Chao H, Walsh CE. Induction of tolerance to human factor VIII in mice. Blood 2001; 97 (10) 3311-2.
    CrossrefPubMedGoogle Scholar
  • 17 Reipert BM, Ahmad RU, Turecek PL, Schwarz HP. Characterization of antibodies induced by human factor VIII in a murine knockout model of hemophilia A. Thromb Haemost 2000; 84 (05) 826-32.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 18 Wu H, Reding M, Qian J, Okita DK, Parker E, Lollar P, Hoyer LW, Conti-Fine BM. Mechanism of the immune response to human factor VIII in murine hemophilia A. Thromb Haemost 2001; 85 (01) 125-33.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 19 Barrow RT, Healey JF, Jacquemin MG, Saint-Remy JM, Lollar P. Antigenicity of putative phospholipid membrane-binding residues in factor VIII. Blood 2001; 97 (01) 169-74.
    CrossrefPubMedGoogle Scholar
  • 20 Horton RM, Cai ZL, Ho SN, Pease LR. Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. Biotechniques 1990; 08 (05) 528-35.
    PubMedGoogle Scholar
  • 21 Seidah NG, Chretien M. Eukaryotic protein processing: endoproteolysis of precursor proteins. Curr Opin Biotechnol 1997; 08 (05) 602-7.
    CrossrefPubMedGoogle Scholar
  • 22 Lind P, Larsson K, Spira J, Sydow-Backman M, Almstedt A, Gray E, Sandberg H. Novel forms of B-domain-deleted recombinant factor VIII molecules Construction and biochemical characterization. Eur J Biochem 1995; 232 (01) 19-27.
    CrossrefPubMedGoogle Scholar
  • 23 Healey JF, Barrow RT, Tamim HM, Lubin IM, Shima M, Scandella D, Lollar P. Residues Glu2181-Val2243 contain a major determinant of the inhibitory epitope in the C2 domain of human factor VIII. Blood 1998; 92 (10) 3701-9.
    PubMedGoogle Scholar
  • 24 Elder B, Lakich D, Gitschier J. Sequence of the murine factor VIII cDNA. Genomics 1993; 16 (02) 374-9.
    CrossrefPubMedGoogle Scholar
  • 25 Funk WD, MacGillivray RT, Mason AB, Brown SA, Woodworth RC. Expression of the aminoerminal half-molecule of human serum transferrin in cultured cells and characterization of the recombinant protein. Biochemistry 1990; 29 (06) 1654-60.
    CrossrefPubMedGoogle Scholar
  • 26 Barrow RT, Healey JF, Gailani D, Scandella D, Lollar P. Reduction of the antigenicity of factor VIII toward complex inhibitory antibody plasmas using multiply-substituted hybrid human/porcine factor VIII molecules. Blood 2000; 95 (02) 564-8.
    PubMedGoogle Scholar
  • 27 Kasper CK, Aledort L, Aronson D, Counts R, Edson JR, van Eys J, Fratantoni J, Green D, Hampton J, Hilgartner M, Levine P, Lazerson J, McMillan C, Penner J, Shapiro S, Shulman NR. Proceedings: A more uniform measurement of factor VIII inhibitors. Thromb Diath Haemorrh 1975; 34 (02) 612.
    PubMedGoogle Scholar
  • 28 Lollar P, Parker ET, Fay PJ. Coagulant properties of hybrid human/porcine factor VIII molecules. J Biol Chem 1992; 267 (33) 23652-7.
    PubMedGoogle Scholar
  • 29 Lubin IM, Healey JF, Scandella D, Runge MS, Lollar P. Elimination of a major inhibitor epitope in factor VIII. J Biol Chem 1994; 269 (12) 8639-41.
    PubMedGoogle Scholar
  • 30 Pace CN, Vajdos F, Fee L, Grimsley G, Gray T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci 1995; 04 (11) 2411-23.
    CrossrefPubMedGoogle Scholar
  • 31 Fay PJ, Haidaris PJ, Smudzin TM. Human factor VIIIa subunit structure Reconstruction of factor VIIIa from the isolated A1/A3-C1-C2 dimer and A2 subunit. J Biol Chem 1991; 266 (14) 8957-62.
    PubMedGoogle Scholar
  • 32 Lamphear BJ, Fay PJ. Factor IXa enhances reconstitution of factor VIIIa from isolated A2 subunit and A1/A3-C1-C2 dimer. J Biol Chem 1992; 267 (06) 3725-30.
    PubMedGoogle Scholar
  • 33 Lollar P, Parker CG. pH-dependent denaturation of thrombin-activated porcine factor VIII. J Biol Chem 1990; 265 (03) 1688-92.
    PubMedGoogle Scholar
  • 34 Lollar P, Parker ET. Structural basis for the decreased procoagulant activity of human factor VIII compared to the porcine homolog. J Biol Chem 1991; 266 (19) 12481-6.
    PubMedGoogle Scholar
  • 35 Healey JF, Lubin IM, Lollar P. The cDNA and derived amino acid sequence of porcine factor VIII. Blood 1996; 88 (11) 4209-14.
    PubMedGoogle Scholar
  • 36 Hoeben RC, Fallaux FJ, Cramer SJ, van den Wollenberg DJ, van Ormondt H, Briet E, van der Eb AJ. Expression of the blood-clotting factor-VIII cDNA is repressed by a transcriptional silencer located in its coding region. Blood 1995; 85 (09) 2447-54.
    PubMedGoogle Scholar
  • 37 Koeberl DD, Halbert CL, Krumm A, Miller AD. Sequences within the coding regions of clotting factor VIII and CFTR block transcriptional elongation. Hum Gene Ther 1995; 06 (04) 469-79.
    CrossrefPubMedGoogle Scholar
  • 38 Fang X, Chen C, Wang Q, Gu J, Chi C. The interaction of the calcium and integrin-binding protein (cibp) with the coagulation factor VIII. Thromb Res 2001; 102 (02) 177-85.
    CrossrefPubMedGoogle Scholar
  • 39 Pipe SW, Morris JA, Shah J, Kaufman RJ. Differential interaction of coagulation factor VIII and factor V with protein chaperones calnexin and calreticulin. J Biol Chem 1998; 273 (14) 8537-44.
    CrossrefPubMedGoogle Scholar
  • 40 Tagliavacca L, Wang Q, Kaufman RJ. ATP-dependent dissociation of nondisulfide-linked aggregates of coagulation factor VIII is a rate-limiting step for secretion. Biochemistry 2000; 39 (08) 1973-81.
    CrossrefPubMedGoogle Scholar
  • 41 Kaufman RJ, Wasley LC, Davies MV, Wise RJ, Israel DI, Dorner AJ. Effect of von Willebrand factor coexpression on the synthesis and secretion of factor VIII in Chinese hamster ovary cells. Mol Cell Biol 1989; 09 (03) 1233-42.
    CrossrefPubMedGoogle Scholar
  • 42 Kaufman RJ, Wasley LC, Dorner AJ. Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells. J Biol Chem 1988; 263 (13) 6352-62.
    PubMedGoogle Scholar
  • 43 Eaton DL, Wood WI, Eaton D, Hass PE, Hollingshead P, Wion K, Mather J, Lawn RM, Vehar GA, Gorman C. Construction and characterization of an active factor VIII variant lacking the central one-third of the molecule. Biochemistry 1986; 25 (26) 8343-7.
    CrossrefPubMedGoogle Scholar
  • 44 Toole JJ, Pittman DD, Orr EC, Murtha P, Wasley LC, Kaufman RJ. A arge region (approximately equalto 95 kDa) of human factor VIII is dispensable for in vitro procoagulant activity. Proc Natl Acad Sci USA 1986; 83 (16) 5939-42.
    CrossrefPubMedGoogle Scholar
  • 45 Swaroop M, Moussalli M, Pipe SW, Kaufman RJ. Mutagenesis of a potential immunoglobulin-binding protein-binding site enhances secretion of coagulation factor VIII. J Biol Chem 1997; 272 (39) 24121-4.
    CrossrefPubMedGoogle Scholar
  • 46 Kaufman RJ, Pipe SW, Tagliavacca L, Swaroop M, Moussalli M. Biosynthesis, assembly and secretion of coagulation factor VIII. Blood Coagul Fibrinolysis 1997; 08 (Suppl. 02) S3-14.
    PubMedGoogle Scholar
  • 47 Baron U, Bujard H. Tet repressor-based system for regulated gene expression in eukaryotic cells: principles and advances. Methods Enzymol 2000; 327: 401-21.
    PubMedGoogle Scholar
  • 48 Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 1992; 89 (12) 5547-51.
    CrossrefPubMedGoogle Scholar
  • 49 Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, Bujard H. Transcriptional activation by tetracyclines in mammalian cells. Science 1995; 268 5218 1766-9.
    CrossrefPubMedGoogle Scholar
  • 50 Hollestelle MJ, Thinnes T, Crain K, Stiko A, Kruijt JK, van Berkel TJ, Loskutoff DJ, van Mourik JA. Tissue distribution of factor VIII gene expression in vivo – a closer look. Thromb Haemost 2001; 86 (03) 855-61.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 51 Lundblad RL, Kingdon HS, Mann KG, White GC. Issues with the assay of factor VIII activity in plasma and factor VIII concentrates. Thromb Haemost 2000; 84 (06) 942-8.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 52 Curtis JE, Helgerson SL, Parker ET, Lollar P. Isolation and characterization of thrombin-activated human factor VIII. J Biol Chem 1994; 269 (08) 6246-51.
    PubMedGoogle Scholar
  • 53 Lollar P, Parker CG. Subunit structure of thrombin-activated porcine factor VIII. Biochemistry 1989; 28 (02) 666-74.
    CrossrefPubMedGoogle Scholar
  • 54 Pipe SW, Eickhorst AN, McKinley SH, Saenko EL, Kaufman RJ. Mild hemophilia A caused by increased rate of factor VIII A2 subunit dissociation: evidence for nonproteolytic inactivation of factor VIIIa in vivo. Blood 1999; 93 (01) 176-83.
    PubMedGoogle Scholar
  • 55 Qian J, Burkly LC, Smith EP, Ferrant JL, Hoyer LW, Scott DW, Haudenschild CC. Role of CD154 in the secondary immune response: the reduction of preexisting splenic germinal centers and anti-factor VIII inhibitor titer. Eur J Immunol 2000; 30 (09) 2548-54.
    CrossrefPubMedGoogle Scholar