Download PDF
Thromb Haemost 2003; 90(03): 385-397
DOI: 10.1160/TH02-09-0086
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

Anti-heavy-chain monoclonal antibodies directed to the acidic regions of the factor VIII molecule inhibit the binding of factor VIII to phospholipids and von Willebrand factor

Sanjeev Raut
1   Division of Haematology, National Institute for Biological Standards and Control, Hertfordshire, UK
,
Sylvie Villard
2   UMR-CNRS, Montpellier, France, University of Leuven, Leuven, Belgium
,
Sabrina Grailly
3   Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
,
Jean-Guy G. Gilles
3   Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
,
Claude Granier
2   UMR-CNRS, Montpellier, France, University of Leuven, Leuven, Belgium
,
Jean-Marie R. Saint-Remy
3   Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
,
Trevor W. Barrowcliffe
1   Division of Haematology, National Institute for Biological Standards and Control, Hertfordshire, UK
› Author Affiliations
Further Information

Publication History

Received 17 September 2002

Accepted after 09 June 2003

Publication Date:
05 December 2017 (online)

    Permissions and Reprints

Summary

Recent studies have shown that inhibitors develop against acidic regions of the FVIII molecule, which contain important functional sites. However, their mechanisms of inhibition are not well understood. In this study, two anti-human FVIII mouse monoclonal antibodies (MAbs), directed towards the exposed acidic regions of the FVIII molecule, were developed, characterised and their mechanisms of inhibition investigated. The two MAbs, F7B4 and F26F6, had inhibitory titres of 32 and 944 BU/mg respectively, had high affinities for the FVIII molecule (KD ~ nM range) and recognised sequences V357-F360 on the acidic a1 region and E724-L731 on the acidic a2 region of the FVIII heavy-chain (HC), respectively. F7B4 inhibited the rate of FXa generation by activated FVIII, whilst both antibodies inhibited FVIII activation by thrombin and blocked thrombin cleavage of FVIII. Furthermore, F7B4 and F26F6 inhibited FVIII binding to (a) phospholipids (IC50: 77 nM and 40 nM respectively), and (b) VWF (IC50: 93 nM and 267 nM respectively), despite both having HC specificity. Experiments with F(ab’)2 fragments confirmed the above findings. Taken together these data represent novel findings in that anti-acidic HC antibodies can inhibit FVIII function by a variety of mechanisms, in particular by interfering with the binding of FVIII to phospholipids & VWF.

Keywords

Factor VIII - monoclonal antibody - epitope - phospholipid - VWF
 

  • References

  • 1 Lusher JM, Arkin S, Abildgaard CF, Schwartz RS. Kogenate Previously Untreated Patient Study Group. Recombinant factor VIII for the treatment of previously untreated patients with haemophilia A. New Eng J Med 1993; 238: 453-9.
    PubMedGoogle Scholar
  • 2 Mannucci PM, Giangrande PLF. Acquired disorders of coagulation. In: Haemostasis and Thrombosis, 3rd Edition. Churchill Livingstone 1994; 949-68.
    PubMedGoogle Scholar
  • 3 Peerlinck K, Arnout J, Giambattista MD. et al. FVIII inhibitors in previously treated haemophilia A patients with a double virus-inactivated plasma derived factor VIII concentrate. Thromb Haemost 1997; 77: 80-6.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 4 Raut S, Di Giambattista M, Bevan SA. et al. Modification of FVIII in therapeutic concentrates after virus inactivation by solvent-detergent and pasteurisation. Thromb Haemost 1998; 80: 624-31.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 5 Bray GL, Gomperts ED, Courter S. et al. the Recombinate Study Group. A multicentre study of recombinant factor VIII (Recombinate): safety, efficacy and inhibitor risk in previously untreated patients with haemophilia A. Blood 1994; 83: 2428-35.
    PubMedGoogle Scholar
  • 6 Arai M, Scandella D, Hoyer LW. Molecular basis of factor VIII inhibition by human antibodies. Antibodies that bind to the factor VIII light chain prevent the interaction of factor VIII with phospholipid. J Clin Invest 1989; 83: 1978-84.
    CrossrefPubMedGoogle Scholar
  • 7 Shima M, Scandella D, Yoshioka A. et al. A factor VIII neutralizing monoclonal antibody and a human inhibitor alloantibody recognizing epitopes in the C2 domain inhibit factor VIII binding to von Willebrand factor and to phosphatidylserine. Thromb Haemost 1993; 69: 240-6.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 8 Lenting PJ, Van de Loo JWP, Donath MJSH. et al. The sequence Glu1811-Lys1818 of human blood coagulation factor VIII comprises a binding site for activated factor IX. J Biol Chem 1996; 271: 1935-40.
    CrossrefPubMedGoogle Scholar
  • 9 Zhong D, Saenko EL, Shima M. et al. Some human inhibitor antibodies interfere with factor VIII binding to factor IX. Blood 1998; 92: 136-42.
    PubMedGoogle Scholar
  • 10 Fijnvandraat K, Celie PH, Turenhout EAM. et al. A human alloantibody interferes with binding of factor IXa to the factor VIII light chain. Blood 1998; 91: 2347-52.
    PubMedGoogle Scholar
  • 11 Lollar P, Parker ET, Curtis JE. et al. Inhibition of human factor VIIIa by anti-A2 subunit antibodies. J Clin Invest 1994; 93: 2497-504.
    CrossrefPubMedGoogle Scholar
  • 12 Fay PJ, Scandella D. Human inhibitor antibodies specific for the factor VIII A2 domain disrupt the interaction between the subunit and factor IXa. J Biol Chem 1999; 274: 29826-30.
    CrossrefPubMedGoogle Scholar
  • 13 Lacroix-Desmazes S, Moreau A, Sooryanarayana-Bonnemain C. et al. Catalytic activity of antibodies against factor VIII in patients with hemophilia A. Nat Med 1999; 5: 1044-7.
    CrossrefPubMedGoogle Scholar
  • 14 Kasper CK, Aledort LM, Counts RB. et al. A more uniform measurement of factor VIII inhibitors. Thromb Diath Haemorrhag 1975; 34: 869-72.
    PubMedGoogle Scholar
  • 15 Foster PA, Fulcher CA, Houghten RA. et al. Localization of the binding regions of a murine monoclonal anti-factor VIII antibody and a human anti-factor VIII alloantibody, both of which inhibit factor VIII procoagulant activity, to amino acid residues threonine351-serine365 of the factor VIII heavy chain. J Clin Invest 1988; 82: 123-8.
    CrossrefPubMedGoogle Scholar
  • 16 Scandella D, Gilbert GE, Shima M. et al. Some factor VIII inhibitor antibodies recognize a common epitope corresponding to C2 domain amino acids 2248 through 2312, which overlap a phospholipid-binding site. Blood 1995; 86: 1811-9.
    PubMedGoogle Scholar
  • 17 Prescott R, Nakai H, Saenko EL. et al. The inhibitor antibody response is more complex in hemophilia A patients than in most nonhemophiliacs with factor VIII autoantibodies. Blood 1997; 89: 3663-71.
    PubMedGoogle Scholar
  • 18 Foster PA, Fulcher CA, Houghten RA. et al. An immunogenic region within the val1670-glu1684 of the factor VIII light chain induces antibodies which inhibit binding of factor VIII to von Willebrand Factor. J Biol Chem 1988; 263: 5230-4.
    PubMedGoogle Scholar
  • 19 Barrow RT, Healey JF, Gailani D. et al. Reduction of the antigenicity of factor VIII toward complex inhibitory antibody plasmas using multiply-substituted hybrid human/porcine factor VIII molecules. Blood 2000; 95: 564-8.
    PubMedGoogle Scholar
  • 20 van den Brink EN, Turenhout EAM, Bank CMC. et al. Molecular analysis of human anti-factor VIII antibodies by V gene phage display identifies a new epitope in the acidic region following the A2 domain. Blood 2000; 96: 540-5.
    PubMedGoogle Scholar
  • 21 Pemberton S, Lindley P, Zaitsev V. et al. Molecular model for the triplicated A domains of human factor VIII based on the crystal structure of human ceruloplasmin. Blood 1997; 89: 2413-21.
    PubMedGoogle Scholar
  • 22 Kemball-Cook G, Tuddenham EG, Wacey AI.. The factor VIII structure and mutation resource site: HAMSTeRS version 4. Nucleic Acids Res 1998; 26 (01) 216-9.
    CrossrefPubMedGoogle Scholar
  • 23 Stoilova-McPhie S, Villoutreix BO, Mertens K. et al. 3-Dimensional structure of membrane-bound coagulation factor VIII: modelling of the factor VIII heterodimer within a 3-dimensional density map derived by electron crystallography. Blood 2002; 99: 1215-23.
    CrossrefPubMedGoogle Scholar
  • 24 Gilles JG, Arnout J, Vermylen J. et al. Anti-factor VIII antibodies of hemophiliac patients are frequently directed towards nonfunctional determinants and do not exhibit isotypic restriction. Blood 1993; 82: 2452-61.
    PubMedGoogle Scholar
  • 25 Lamoyi E. Preparation of F(ab’)2 fragments from mouse IgG of various subclasses. Methods Enzymol 1986; 121: 652-63.
    PubMedGoogle Scholar
  • 26 Raut S, Gaffney PJ. Evaluation of the fibrin binding profile of two anti-fibrin monoclonal antibodies. Thromb Haemost 1996; 76: 56-64.
    PubMedGoogle Scholar
  • 27 Schagger H, von Jagow G. Tricine-sodium dodecyl sulphate polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 1987; 166: 368-79.
    CrossrefPubMedGoogle Scholar
  • 28 Mattson DL, Bellehumeur TG. Comparison of three chemiluminescent horseradish peroxidase substrates for immunoblotting. Anal Biochem 1996; 240 (02) 306-8.
    CrossrefPubMedGoogle Scholar
  • 29 Jacquemin M, Benhida A, Peerlinck K. et al. A human antibody directed to the factor VIII C1 domain inhibits factor VIII cofactor activity and binding to von Willebrand factor. Blood 2000; 95: 156-63.
    PubMedGoogle Scholar
  • 30 Jacquemin MG, Desqueper BG, Benhida A. et al. Mechanism and kinetics of factor VIII inactivation: study with an IgG4 monoclonal antibody derived from a hemophilia A patient with inhibitor. Blood 1998; 92: 496-506.
    PubMedGoogle Scholar
  • 31 Frank R. Spot Synthesis: An easy technique for the positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedron 1992; 48: 9217-32.
    CrossrefPubMedGoogle Scholar
  • 32 Molina F, Laune D, Gougat C. et al. Improved performances of spot multiple peptide synthesis. Peptide Res 1996; 9: 151-5.
    PubMedGoogle Scholar
  • 33 Gausepohl H, Boulin C, Kraft M. et al. Automated multiple peptide synthesis. Peptide Res 1992; 5: 315-20.
    PubMedGoogle Scholar
  • 34 Verbruggen B, Novakova I, Wessels H. et al. The Nijmegen modification of the Bethesda Assay for factor VIII: C inhibitors: Improved specificity and reliability. Thromb Haemost 1995; 73: 247-51.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 35 Sandberg H, Almstedt A, Brandt J. et al. Structural and functional characteristics of the B-domain-deleted recombinant factor VIII protein, r-VIII SQ. Thromb Haemost 2001; 85 (01) 93-100.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 36 Hubbard AR, Weller L, Bevan SA. Activation profiles of FVIII in concentrates reflect onestage/chromogenic potency discrepancies. Br J Haematol 2002; 117: 957-60.
    CrossrefPubMedGoogle Scholar
  • 37 Kemball-Cook G, Tubbs JE, Dawson NJ. et al. The behaviour of different factor VIII concentrates in a chromogenic factor X-activating system. Br J Haematol 1993; 84: 273-8.
    CrossrefPubMedGoogle Scholar
  • 38 Bloom JW. The interaction of rDNA factor VIII, factordes-797-1562 and factordes-797-1562-derived peptides with phospholipid. Thromb Res 1987; 48: 439-48.
    CrossrefPubMedGoogle Scholar
  • 39 Raut S, Weller L, Barrowcliffe TW. Phospholipid binding of factor VIII in different therapeutic concentrates. Br J Haematol 1999; 107: 323-9.
    CrossrefPubMedGoogle Scholar
  • 40 Fulcher CA, Roberts JR, Holland LZ. et al. Human factor VIII procoagulant protein. Monoclonal antibodies define precursor-product relationships and functional epitopes. J Clin Invest 1985; 76: 117-24.
    CrossrefPubMedGoogle Scholar
  • 41 Vehar GA, Keyt B, Eaton D. et-al Structure of human factor VIII. Nature 1984; 312: 337-42.
    CrossrefPubMedGoogle Scholar
  • 42 Wood WI, Capon DJ, Simonsen CC. et al. Expression of active human factor VIII from recombinant DNA clones. Nature 1984; 312: 330-7.
    CrossrefPubMedGoogle Scholar
  • 43 Fay PJ, Haidaris PJ, Huggins CF. Role of the COOH-terminal acidic region of A1 subunit in A2 subunit retention in human factor VIIIa. J Biol Chem 1993; 268: 17861-6.
    PubMedGoogle Scholar
  • 44 Lapan KA, Fay PJ. Localization of a factor X interactive site in the A1 subunit of factor VIIIa. J Biol Chem 1997; 272: 2082-8.
    CrossrefPubMedGoogle Scholar
  • 45 Foster PA, Fulcher CA, Houghten RA. et al. Synthetic factor VIII peptides with amino acid sequences contained within the C2 domain of factor VIII inhibit factor VIII binding to phosphatidylserine. Blood 1990; 75 (10) 1999-2004.
    PubMedGoogle Scholar
  • 46 Stoylova SS, Lenting PJ, Kemball-Cook G. et al. Electron crystallography of human blood coagulation factor VIII bound to phospholipid monolayers. J Biol Chem 1999; 274: 36573-8.
    CrossrefPubMedGoogle Scholar
  • 47 Pittman DD, Kaufman RJ. Proteolytic requirements for thrombin activation of anti-hemophilic factor (factor VIII). Proc Natl Acad Sci USA 1988; 85: 2429-33.
    CrossrefPubMedGoogle Scholar
  • 48 Donath MJ, Lenting PJ, Van-Mourik JA. et al. Kinetics of factor VIII light-chain cleavage by thrombin and factor Xa. A regulatory role of the factor VIII heavy-chain region Lys713-Arg740. Eur J Biochem 1996; 240: 365-72.
    CrossrefPubMedGoogle Scholar
  • 49 Gensana M, Altisent C, Aznar JA. et al. Influence of von Willebrand factor on the reactivity of human factor VIII inhibitors with factor VIII. Haemophilia 2001; 7 (04) 369-74.
    CrossrefPubMedGoogle Scholar
  • 50 Hamer RJ, Koedam JA, Beeser-Visser NH. et al. The effect of thrombin on the complex between factor VIII and von Willebrand factor. Eur J Biochem 1987; 167 (02) 253-9.
    CrossrefPubMedGoogle Scholar
  • 51 Fay PJ. Reconstitution of human factor VIII from isolated subunits. Arch Biochem Biophys 1988; 262 (02) 525-31.
    CrossrefPubMedGoogle Scholar