In silico and in vitro evaluation of the impact of mutations in non-severe haemophilia A patients on assay discrepancies

  • Behnaz PezeshkpoorEmail author
  • M. Gazorpak
  • A-C. Berkemeier
  • H. Singer
  • A. Pavlova
  • A. Biswas
  • J. Oldenburg
Original Article


Haemophilia A (HA) is caused by a lack or reduced amount of factor VIII protein (FVIII). About one-third of patients with non-severe HA carrying specific missense mutations show discrepant results between FVIII activity (FVIII:C), measured by one-stage or chromogenic two-stage assays. The aim of this study was to elucidate the mechanism underlying the assay discrepancy in vitro and in silico. Thirteen missense mutations in the Factor 8-gene associated with discrepant results in patients were transiently expressed. FVIII:C of the mutations was determined using two one-stage assays (FVIII:C1st, FVIII:CBonn) and a two-stage chromogenic assay (FVIII:Cchr). Furthermore, thrombin generation test (TGT) and in silico analysis were performed to investigate the haemostatic potential as well as the structural impact of the variants, respectively. For the majority (9/13) of the analysed mutations, the discrepancy was confirmed. Moreover, we established a modified TGT protocol for in vitro characterization of FVIII. Hence, TGT parameters were significantly impaired in the group of variants associated with higher chromogenic values. Additionally, in silico analysis revealed the impact of the mutations on FVIII protein structure leading to assay discrepancy. Moreover, the data shows that also among one-stage clotting assays, assay discrepancy is observed. Our results show that for the majority of mutations, application of a global assay like TGT method could help to improve diagnosis or correct assessment of the severity of HA.


FVIII:C One-stage assay Chromogenic assay Discrepancy Haemophilia A 


Author contributions

BP, JO and MG designed the study; MG and ACB performed research; BP and MG analysed the data; AB performed in silico analysis; AP and JO revised the manuscript; and BP and MG wrote the paper.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Supplementary material

277_2019_3691_MOESM1_ESM.pdf (662 kb)
ESM 1 (PDF 661 kb)


  1. 1.
    Oldenburg J, Ananyeva NM, Saenko EL (2004) Molecular basis of haemophilia A. Haemophilia 10(Suppl 4):133–139. CrossRefGoogle Scholar
  2. 2.
    Oldenburg J, Brackmann HH, Hanfland P, Schwaab R (2000) Molecular genetics in haemophilia A. Vox Sang 78(Suppl 2):33–38Google Scholar
  3. 3.
    Pezeshkpoor B, Pavlova A, Oldenburg J, El-Maarri O (2014) F8 genetic analysis strategies when standard approaches fail. Hamostaseologie 34(2):167–173. CrossRefGoogle Scholar
  4. 4.
    de Brasi C, El-Maarri O, Perry DJ, Oldenburg J, Pezeshkpoor B, Goodeve A (2014) Genetic testing in bleeding disorders. Haemophilia 20(Suppl 4):54–58. CrossRefGoogle Scholar
  5. 5.
    Langdell RD, Wagner RH, Brinkhous KM (1953) Effect of antihemophilic factor on one-stage clotting tests; a presumptive test for hemophilia and a simple one-stage antihemophilic factor assay procedure. J Lab Clin Med 41(4):637–647Google Scholar
  6. 6.
    Oldenburg J, Pavlova A (2010) Discrepancy between one-stage and chromogenic factor VIII activity assay results can lead to misdiagnosis of haemophilia A phenotype. Hamostaseologie 30(4):207–211CrossRefGoogle Scholar
  7. 7.
    Potgieter JJ, Damgaard M, Hillarp A (2015) One-stage vs. chromogenic assays in haemophilia A. Eur J Haematol 94 Suppl 77:38–44. CrossRefGoogle Scholar
  8. 8.
    Biggs R (1955) Assessment of clotting efficiency. Br Med Bull 11(1):5–10CrossRefGoogle Scholar
  9. 9.
    Biggs R, Eveling J, Richards G (1955) The assay of antihaemophilic-globulin activity. Br J Haematol 1(1):20–34CrossRefGoogle Scholar
  10. 10.
    Barrowcliffe TW, Raut S, Sands D, Hubbard AR (2002) Coagulation and chromogenic assays of factor VIII activity: general aspects, standardization, and recommendations. Semin Thromb Hemost 28(3):247–256. CrossRefGoogle Scholar
  11. 11.
    Schwaab R, Oldenburg J, Kemball-Cook G, Albert T, Juhler C, Hanfland P, Ingerslev J (2000) Assay discrepancy in mild haemophilia A due to a factor VIII missense mutation (Asn694Ile) in a large Danish family. Br J Haematol 109(3):523–528CrossRefGoogle Scholar
  12. 12.
    Trossaert M, Lienhart A, Nougier C, Fretigny M, Sigaud M, Meunier S, Fouassier M, Ternisien C, Negrier C, Dargaud Y (2014) Diagnosis and management challenges in patients with mild haemophilia A and discrepant FVIII measurements. Haemophilia 20(4):550–558. CrossRefGoogle Scholar
  13. 13.
    Hakeos WH, Miao H, Sirachainan N, Kemball-Cook G, Saenko EL, Kaufman RJ, Pipe SW (2002) Hemophilia A mutations within the factor VIII A2-A3 subunit interface destabilize factor VIIIa and cause one-stage/two-stage activity discrepancy. Thromb Haemost 88(5):781–787. CrossRefGoogle Scholar
  14. 14.
    Mumford AD, Laffan M, O'Donnell J, McVey JH, Johnson DJ, Manning RA, Kemball-Cook G (2002) A Tyr346-->Cys substitution in the interdomain acidic region a1 of factor VIII in an individual with factor VIII:C assay discrepancy. Br J Haematol 118(2):589–594CrossRefGoogle Scholar
  15. 15.
    Provaznikova D, Houskova K, Radovska A, Salaj P, Hrachovinova I (2015) Novel mutations associated with a discrepancy between one-stage and chromogenic FVIII activity assays. Haemophilia 21(4):e330–e332. CrossRefGoogle Scholar
  16. 16.
    Pavlova A, Delev D, Pezeshkpoor B, Muller J, Oldenburg J (2014) Haemophilia A mutations in patients with non-severe phenotype associated with a discrepancy between one-stage and chromogenic factor VIII activity assays. Thromb Haemost 111(5):851–861. CrossRefGoogle Scholar
  17. 17.
    Pezeshkpoor B, Schreck U, Biswas A, Driesen J, Berkemeier AC, Pavlova A, Muller J, Oldenburg J (2017) An in silico and in vitro approach to elucidate the impact of residues flanking the cleavage scissile bonds of FVIII. PLoS One 12(7):e0180456. CrossRefGoogle Scholar
  18. 18.
    Hemker HC, Giesen P, Al Dieri R, Regnault V, de Smedt E, Wagenvoord R, Lecompte T, Beguin S (2003) Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb 33(1):4–15CrossRefGoogle Scholar
  19. 19.
    Venkateswarlu D (2010) Structural investigation of zymogenic and activated forms of human blood coagulation factor VIII: a computational molecular dynamics study. BMC Struct Biol 10:7. CrossRefGoogle Scholar
  20. 20.
    Ngo JC, Huang M, Roth DA, Furie BC, Furie B (2008) Crystal structure of human factor VIII: implications for the formation of the factor IXa-factor VIIIa complex. Structure 16(4):597–606. CrossRefGoogle Scholar
  21. 21.
    Shen BW, Spiegel PC, Chang CH, Huh JW, Lee JS, Kim J, Kim YH, Stoddard BL (2008) The tertiary structure and domain organization of coagulation factor VIII. Blood 111(3):1240–1247. CrossRefGoogle Scholar
  22. 22.
    Krieger E, Vriend G (2014) YASARA view - molecular graphics for all devices - from smartphones to workstations. Bioinformatics 30(20):2981–2982. CrossRefGoogle Scholar
  23. 23.
    Backes BJ, Harris JL, Leonetti F, Craik CS, Ellman JA (2000) Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin. Nat Biotechnol 18(2):187–193. CrossRefGoogle Scholar
  24. 24.
    Petrassi HM, Williams JA, Li J, Tumanut C, Ek J, Nakai T, Masick B, Backes BJ, Harris JL (2005) A strategy to profile prime and non-prime proteolytic substrate specificity. Bioorg Med Chem Lett 15(12):3162–3166. CrossRefGoogle Scholar
  25. 25.
    Selvaraj SR, Scheller AN, Miao HZ, Kaufman RJ, Pipe SW (2012) Bioengineering of coagulation factor VIII for efficient expression through elimination of a dispensable disulfide loop. J Thromb Haemost 10(1):107–115. CrossRefGoogle Scholar
  26. 26.
    Kihlberg K, Strandberg K, Rosen S, Ljung R, Astermark J (2017) Discrepancies between the one-stage clotting assay and the chromogenic assay in haemophilia B. Haemophilia 23(4):620–627. CrossRefGoogle Scholar
  27. 27.
    Pemberton S, Lindley P, Zaitsev V, Card G, Tuddenham EG, Kemball-Cook G (1997) A molecular model for the triplicated A domains of human factor VIII based on the crystal structure of human ceruloplasmin. Blood 89(7):2413–2421Google Scholar
  28. 28.
    Keeling DM, Sukhu K, Kemball-Cook G, Waseem N, Bagnall R, Lloyd JV (1999) Diagnostic importance of the two-stage factor VIII:C assay demonstrated by a case of mild haemophilia associated with His1954-->Leu substitution in the factor VIII A3 domain. Br J Haematol 105(4):1123–1126CrossRefGoogle Scholar
  29. 29.
    Pipe SW, Eickhorst AN, McKinley SH, Saenko EL, Kaufman RJ (1999) Mild hemophilia A caused by increased rate of factor VIII A2 subunit dissociation: evidence for nonproteolytic inactivation of factor VIIIa in vivo. Blood 93(1):176–183Google Scholar
  30. 30.
    Wakabayashi H, Griffiths AE, Fay PJ (2009) Combining mutations of charged residues at the A2 domain interface enhances factor VIII stability over single point mutations. J Thromb Haemost 7(3):438–444. CrossRefGoogle Scholar
  31. 31.
    Bloem E, Meems H, van den Biggelaar M, Mertens K, Meijer AB (2013) A3 domain region 1803-1818 contributes to the stability of activated factor VIII and includes a binding site for activated factor IX. J Biol Chem 288(36):26105–26111. CrossRefGoogle Scholar
  32. 32.
    Venkateswarlu D (2014) Structural insights into the interaction of blood coagulation co-factor VIIIa with factor IXa: a computational protein-protein docking and molecular dynamics refinement study. Biochem Biophys Res Commun 452(3):408–414. CrossRefGoogle Scholar
  33. 33.
    Schwaab R, Pavlova A, Albert T, Caspers M, Oldenburg J (2013) Significance of F8 missense mutations with respect to inhibitor formation. Thromb Haemost 109(3):464–470. CrossRefGoogle Scholar
  34. 34.
    Makris M, Peyvandi F (2014) Assaying FVIII activity: one method is not enough, and never was. Haemophilia 20(3):301–303. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute of Experimental Hematology and Transfusion MedicineUniversity of BonnBonnGermany
  2. 2.Center for Rare Diseases Bonn (ZSEB)University Clinic BonnBonnGermany

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