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Thromb Haemost 1996; 75(01): 036-044
DOI: 10.1055/s-0038-1650218
Original Article
Schattauer GmbH Stuttgart

Effect of Heterologous Factor V Heavy Chain Sequences on the Secretion of Recombinant Human Factor VIII

Thomas L Ortel
1   The Division of Hematology-Oncology, Departments of Medicine and Pathology, Duke University Medical Center, Durham, North Carolina, USA
,
Karen D Moore
1   The Division of Hematology-Oncology, Departments of Medicine and Pathology, Duke University Medical Center, Durham, North Carolina, USA
,
Mirella Ezban
2   The Division of Coagulation Research, Novo-Nordisk A/S, Copenhagen, Denmark
,
William H Kane
1   The Division of Hematology-Oncology, Departments of Medicine and Pathology, Duke University Medical Center, Durham, North Carolina, USA
› Author Affiliations
Further Information

Publication History

Received 02 August 1995

Accepted after revision 03 October 1995

Publication Date:
10 July 2018 (online)

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Summary

Factor VIII and factor V share a repetitive domain structure of A1-A2-B-A3-C1-C2. To define the region(s) within the factor VIII heavy chain that result in inefficient expression of the recombinant protein, we expressed a series of factor VIH/factor V chimeras that contained heterologous sequences from the A1 and/or A2 domains. Substitution of the factor VIIIA1 domain dramatically reduced secretion of factor V ~ 500-fold, whereas substitution of the factor VIII A2 domain had minimal effect on secretion. Conversely, substitution of the factor V A1 domain increased secretion of factor VIII ~3-fold, whereas substitution of the factor V A2 domain actually reduced secretion ~4-fold. Pulse chase experiments confirmed that reduced expression levels were due to decreased secretion rather than instability of secreted protein. Smaller substitutions did not further localize within the A1 domain the regions responsible for inefficient secretion.

 

  • References

  • 1 Kane WH, Davie EW. Blood coagulation factors V and VIII: structural and functional similarities and their relationship to hemorrhagic and thrombotic disorders. Blood 1988; 71: 539-555
    PubMedGoogle Scholar
  • 2 Ortel TL, Keller FG, Kane WH. Factor V. In: Molecular Biology of Thrombosis and Hemostasis High KA, Roberts HR. eds Marcel Dekker; New York: 1995. pp 119-146
  • 3 Eaton D, Rodriguez H, Vehar GA. Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity. Biochemistry 1986; 25: 505-512
    CrossrefPubMedGoogle Scholar
  • 4 Pittman DD, Kaufman RJ. Proteolytic requirements for thrombin activation of anti-hemophilic factor (factor VIII). Proc Natl Acad Sci USA 1988; 85: 2429-2433
    CrossrefPubMedGoogle Scholar
  • 5 Kane WH, Majerus PW. Purification and characterization of human coagulation factor V. J Biol Chem 1981; 256: 1002-1007
    PubMedGoogle Scholar
  • 6 Suzuki K, Dahlback B, Stenflo J. Thrombin-catalyzed activation of human coagulation factor V. J Biol Chem 1982; 257: 6556-6564
    PubMedGoogle Scholar
  • 7 Keller FG, Ortel TL, Quinn-Allen MA, Kane WH. Thrombin-catalyzed activation of recombinant factor V. Biochemistry 1995; 34: 4118-4124
    CrossrefPubMedGoogle Scholar
  • 8 Monkovic DD, Tracy PB. Activation of human factor V by factor Xa and thrombin. Biochemistry 1990; 29: 1118-1128
    CrossrefPubMedGoogle Scholar
  • 9 Kane WH, Devore-Carter D, Ortel TL. Expression and characterization of recombinant human factor V and a mutant lacking a major portion of the connecting region. Biochemistry 1990; 29: 6762-6768
    CrossrefPubMedGoogle Scholar
  • 10 Toole JJ, Pittman DD, Orr EC, Murtha P, Wasley LC, Kaufman RJ. A large region (approximately equal to 95 kDA) of human factor VIII is dispensable for in vitro procoagulant activity. Proc Natl Acad Sci USA 1986; 83: 5939-5942
    CrossrefPubMedGoogle Scholar
  • 11 Meulien P, Faure T, Mischler F, Harrer H, Ulrich P, Bouderbala B, Dott K, Sainte Marie M, Mazurier C, Wiesel J-P, Van de Pol H, Cazenave J-P, Courtney M, Pavirani A. A new recombinant procoagulant protein derived from the cDNA encoding human factor VIII. Protein Engineering 1995; 2: 301-306
    PubMedGoogle Scholar
  • 12 Kaufman RJ. Recombinant DNA technology for the production of Factor VIII for treatment of hemophilia A. In: Excerpta Medica, Vol. 943. Hemophilia and von Willebrand’s Disease in the 1990s: A new decade of hopes and challenges (Proceeding of the XIX Congress of the World Federation of Hemophilia, Washington, D.C., August 14-19, 1990) Lusher JM, Kessler CM. eds Elsevier Science Publishers B. V; New York: 1991. pp 235-246
  • 13 Lynch CM, Israel DI, Kaufman RJ, Miller D. Sequences in the coding region of clotting factor VIII act as dominant inhibitors of RNA accumulation and protein production. Hum Gene Ther 1994; 4: 259-272
    PubMedGoogle Scholar
  • 14 Pittman DD, Marquette KA, Kaufman RJ. Role of the B domain for factor VIII and factor V expression and function. Blood 1995; 84: 4214-4225
    PubMedGoogle Scholar
  • 15 Hoeben RC, Fallaux FJ, Cramer SJ, van den Wollenberg JM, 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: 2447-2454
    PubMedGoogle Scholar
  • 16 Kaufman RJ, Wasley LC, Domer AJ. Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells. J Biol Chem 1988; 263: 6352-6362
    PubMedGoogle Scholar
  • 17 Domer AJ, Bole DG, Kaufman RJ. The relationship of N-linked glycosyla-tion and heavy chain-binding protein association with the secretion of glycoproteins. J Cell Biol 1987; 105: 2665-2674
    CrossrefPubMedGoogle Scholar
  • 18 Burke RL, Pachl C, Quiroga M, Rosenberg S, Haigwood N, Nordfang O, Ezban M. The functional domains of coagulation factor VIILC. J Biol Chem 1986; 261: 12574-12588
    PubMedGoogle Scholar
  • 19 Ortel TL, Devore-Carter D, Quinn-Allen MA, Kane WH. Deletion analysis of recombinant human factor V. Evidence for a phosphatidylserine binding site in the second C-type domain. J Biol Chem 1992; 267: 4189-4198
    PubMedGoogle Scholar
  • 20 Marquette KA, Pittman DD, Kaufman RJ. A 110-amino acid region within the A1-domain of coagulation factor VIII inhibits secretion from mammalian cells. J Biol Chem 1995; 250: 10297-10303
    PubMedGoogle Scholar
  • 21 Kane WH, Davie EW. Cloning of a cDNA coding for human factor V, a blood coagulation factor homologous to factor VIII and ceruloplasmin. Proc Natl Acad Sci USA 1986; 83: 6800-6804
    CrossrefPubMedGoogle Scholar
  • 22 Ortel TL, Quinn-Alien MA, Keller FG, Peterson JA, Larocca D, Kane WH. Localization of functionally important epitopes within the second C-type domain of coagulation factor V using recombinant chimeras. J Biol Chem 1994; 269: 15898-15905
    CrossrefPubMedGoogle Scholar
  • 23 Mikkelsen J, Thomsen J, Ezban M. Heterogeneity in the tyrosine sulfation of Chinese hamster ovary cell produced recombinant FVIII. Biochemistry 1991; 30: 1533-1537
    CrossrefPubMedGoogle Scholar
  • 24 Harlow E, Lane D. Antibodies: A Laboratory Manual, Cold Spring Harbor. New York: Cold Spring Harbor Laboratory; 1988
    Google Scholar
  • 25 Truett MA, Blacher R, Burke RL, Caput D, Chu D, Dina D, Hartog K, Kuo CH, Masiarz FR, Merryweather JP, Najarian R, Pachl C, Potter SJ, Puma J, Quiroga M, Rail LB, Randolph A, Urdea MS, Valenzuela P, Dahl HH, Favalaro J, Hansen J, Nordfang O, Ezban M. Characterization of the polypeptide composition of human factor VIILC and the nucleotide sequence and expression of the human kidney cDNA. DNA 1985; 4: 333-349
    CrossrefPubMedGoogle Scholar
  • 26 Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 1989; 77: 61-68
    CrossrefPubMedGoogle Scholar
  • 27 Gitschier J, Wood WI, Goralka TM, Wion KL, Chen EY, Eaton DH, Vehar GA, Capon DJ, Lawn RM. Characterization of the human factor VIII gene. Nature 1984; 312: 326-330
    CrossrefPubMedGoogle Scholar
  • 28 Cripe LD, Moore KD, Kane WH. Structure of the gene for human coagulation factor V. Biochemistry 1992; 31: 3777-3785
    CrossrefPubMedGoogle Scholar
  • 29 Nordfang O, Ezban M, Dinesen B. Reactivity of factor VIII inhibitors in a micro ELISA for factor VIII: CAg and in solid phase immunoisolation of VIII: CAg. Thromb Haemost 1985; 53: 346-350
    Article in Thieme ConnectPubMedGoogle Scholar
  • 30 Vehar GA, Keyt B, Eaton DL, Rodriquez H, O’Brien DP, Rotblat F, Oppermann H, Keck R, Wood WI, Harkins RN, Tuddenham EGD, Lawn RM, Capon DJ. Structure of human factor VIII. Nature 1984; 312: 337-342
    CrossrefPubMedGoogle Scholar
  • 31 Kane WH, Ichinose A, Hagen FS, Davie EW. Cloning of cDNAs coding for the heavy chain region and connecting region of human factor V, a blood coagulation factor with four types of internal repeats. Biochemistry 1987; 26: 6508-6514
    CrossrefPubMedGoogle Scholar