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Thromb Haemost 2019; 119(08): 1283-1294
DOI: 10.1055/s-0039-1688907
Coagulation and Fibrinolysis
Georg Thieme Verlag KG Stuttgart · New York

Increased Accumulation and Retention of rhFVIIa (eptacog beta) in Knee Joints of Hemophilia A Mice Compared to Wild-Type Mice

Jhansi Magisetty
1   Department of Cellular and Molecular Biology, The University of Texas Health Science Center at Tyler, Tyler, Texas, United States
,
Usha R. Pendurthi
1   Department of Cellular and Molecular Biology, The University of Texas Health Science Center at Tyler, Tyler, Texas, United States
,
SubbaRao V. Madhunapantula
2   Center of Excellence in Molecular Biology and Regenerative Medicine Laboratory, Department of Biochemistry, JSS Medical College, JSS Academy of Higher Education & Research, Mysuru, Karnataka, India
,
Jerry Grandoni 
3   LFB USA, Framingham, Massachusetts, United States
,
L. Vijaya Mohan Rao
1   Department of Cellular and Molecular Biology, The University of Texas Health Science Center at Tyler, Tyler, Texas, United States
› Author Affiliations Funding This work was supported by grants from HEMA Biologics, LLC and National Heart, Lung, and Blood Institute (HL107483) to L.V.M.R.
Further Information

Publication History

07 September 2018

31 March 2019

Publication Date:
26 May 2019 (online)

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Abstract

Our earlier studies showed that recombinant human factor VIIa (rhFVIIa) administered intravascularly in mice disappeared rapidly from the circulation. However, a small fraction of rhFVIIa that entered extravascular remained functionally active for an extended period. The present study aims to investigate the dose-dependency of rhFVIIa accumulation and retention in mouse knee joints and test whether the hemophilic condition affects rhFVIIa sequestration in joints. Wild-type and FVIII−/− mice were injected with three doses of rhFVIIa (eptacog beta, 90, 250, and 500 μg/kg) via the tail vein. At varying times following rhFVIIa administration, blood and knee joints were collected to measure FVIIa activity and antigen levels in plasma and joint tissues. Joint tissue sections were analyzed by immunohistochemistry for the presence of rhFVIIa. Vascular permeability was assessed by either Evans Blue dye or fluorescein dextran extravasation. The study showed that rhFVIIa accumulated in knee joints of wild-type and FVIII−/− mice in a dose-dependent manner. rhFVIIa antigen and FVIIa activity could be detectable in joints for at least 7 days. Significantly higher levels of rhFVIIa accumulation were observed in knee joints of FVIII−/− mice compared with that of wild-type mice. Immunohistochemical analyses confirmed higher levels of rhFVIIa retention in FVIII−/− mice compared with wild-type mice. Additional studies showed that FVIII−/− mice were more permissible to vascular leakage. In conclusion, the present data demonstrate a dose-dependent accumulation of rhFVIIa in knee joints, and the hemophilic condition enhances the entry of rhFVIIa from circulation to the extravascular. The present data will be useful in improving rhFVIIa prophylaxis.

Keywords

factor VIIa - eptacog beta - hemophilia - permeability - prophylaxis

Authors' Contributions

J.M. performed experiments and analyzed the data. J.G. provided rFVIIa. U.R.P. contributed in designing the study. S.V.M. had an overall supervisory role to J.M. L.V.M.R conceived and designed the research, analyzed the data, and wrote the manuscript. All authors contributed to the preparation of the manuscript.


Supplementary Material

 

  • References

  • 1 Hedner U. Factor VIIa and its potential therapeutic use in bleeding-associated pathologies. Thromb Haemost 2008; 100 (04) 557-562
    Article in Thieme ConnectPubMedGoogle Scholar
  • 2 Abshire T, Kenet G. Recombinant factor VIIa: review of efficacy, dosing regimens and safety in patients with congenital and acquired factor VIII or IX inhibitors. J Thromb Haemost 2004; 2 (06) 899-909
    CrossrefPubMedGoogle Scholar
  • 3 Logan AC, Yank V, Stafford RS. Off-label use of recombinant factor VIIa in U.S. hospitals: analysis of hospital records. Ann Intern Med 2011; 154 (08) 516-522
    PubMedGoogle Scholar
  • 4 Mathew P, Young G. Recombinant factor VIIa in paediatric bleeding disorders--a 2006 review. Haemophilia 2006; 12 (05) 457-472
    CrossrefPubMedGoogle Scholar
  • 5 Witmer CM, Huang YS, Lynch K, Raffini LJ, Shah SS. Off-label recombinant factor VIIa use and thrombosis in children: a multi-center cohort study. J Pediatr 2011; 158 (05) 820-825
    CrossrefPubMedGoogle Scholar
  • 6 Saxon BR, Shanks D, Jory CB, Williams V. Effective prophylaxis with daily recombinant factor VIIa (rFVIIa-Novoseven) in a child with high titre inhibitors and a target joint. Thromb Haemost 2001; 86 (04) 1126-1127
    Article in Thieme ConnectPubMedGoogle Scholar
  • 7 Young G, McDaniel M, Nugent DJ. Prophylactic recombinant factor VIIa in haemophilia patients with inhibitors. Haemophilia 2005; 11 (03) 203-207
    CrossrefPubMedGoogle Scholar
  • 8 Blatny J, Kohlerova S, Zapletal O, Fiamoli V, Penka M, Smith O. Prophylaxis with recombinant factor VIIa for the management of bleeding episodes during immune tolerance treatment in a boy with severe haemophilia A and high-response inhibitors. Haemophilia 2008; 14 (05) 1140-1142
    CrossrefPubMedGoogle Scholar
  • 9 Morfini M, Auerswald G, Kobelt RA. , et al. Prophylactic treatment of haemophilia patients with inhibitors: clinical experience with recombinant factor VIIa in European Haemophilia Centres. Haemophilia 2007; 13 (05) 502-507
    CrossrefPubMedGoogle Scholar
  • 10 Tcheng WY, Donkin J, Konzal S, Wong WY. Recombinant factor VIIa prophylaxis in a patient with severe congenital factor VII deficiency. Haemophilia 2004; 10 (03) 295-298
    CrossrefPubMedGoogle Scholar
  • 11 Mathijssen NC, Masereeuw R, Verbeek K. , et al. Prophylactic effect of recombinant factor VIIa in factor VII deficient patients. Br J Haematol 2004; 125 (04) 494-499
    CrossrefPubMedGoogle Scholar
  • 12 Konkle BA, Ebbesen LS, Erhardtsen E. , et al. Randomized, prospective clinical trial of recombinant factor VIIa for secondary prophylaxis in hemophilia patients with inhibitors. J Thromb Haemost 2007; 5 (09) 1904-1913
    CrossrefPubMedGoogle Scholar
  • 13 Young G, Auerswald G, Jimenez-Yuste V. , et al. PRO-PACT: retrospective observational study on the prophylactic use of recombinant factor VIIa in hemophilia patients with inhibitors. Thromb Res 2012; 130 (06) 864-870
    CrossrefPubMedGoogle Scholar
  • 14 Gopalakrishnan R, Hedner U, Ghosh S. , et al. Bio-distribution of pharmacologically administered recombinant factor VIIa (rFVIIa). J Thromb Haemost 2010; 8 (02) 301-310
    CrossrefPubMedGoogle Scholar
  • 15 Gopalakrishnan R, Hedner U, Clark C, Pendurthi UR, Rao LV. rFVIIa transported from the blood stream into tissues is functionally active. J Thromb Haemost 2010; 8 (10) 2318-2321
    CrossrefPubMedGoogle Scholar
  • 16 Sen P, Clark CA, Gopalakrishnan R. , et al. Factor VIIa binding to endothelial cell protein C receptor: differences between mouse and human systems. Thromb Haemost 2012; 107 (05) 951-961
    Article in Thieme ConnectPubMedGoogle Scholar
  • 17 Clark CA, Vatsyayan R, Hedner U, Esmon CT, Pendurthi UR, Rao LV. Endothelial cell protein C receptor-mediated redistribution and tissue-level accumulation of factor VIIa. J Thromb Haemost 2012; 10 (11) 2383-2391
    CrossrefPubMedGoogle Scholar
  • 18 Vatsyayan R, Kothari H, Mackman N, Pendurthi UR, Rao LV. Inactivation of factor VIIa by antithrombin in vitro, ex vivo and in vivo: role of tissue factor and endothelial cell protein C receptor. PLoS One 2014; 9 (08) e103505
    CrossrefPubMedGoogle Scholar
  • 19 Chevreux G, Faid V, Scohyers JM, Bihoreau N. N-/O-glycosylation analysis of human FVIIa produced in the milk of transgenic rabbits. Glycobiology 2013; 23 (12) 1531-1546
    CrossrefPubMedGoogle Scholar
  • 20 Chevreux G, Tilly N, Leblanc Y. , et al. Biochemical characterization of LR769, a new recombinant factor VIIa bypassing agent produced in the milk of transgenic rabbits. Haemophilia 2017; 23 (04) e324-e334
    CrossrefPubMedGoogle Scholar
  • 21 Grandoni J, Perret G, Forier C. Kinetic analysis and binding studies of a new recombinant human factor VIIa for treatment of haemophilia. Haemophilia 2017; 23 (02) 300-308
    CrossrefPubMedGoogle Scholar
  • 22 Morrissey JH, Macik BG, Neuenschwander PF, Comp PC. Quantitation of activated factor VII levels in plasma using a tissue factor mutant selectively deficient in promoting factor VII activation. Blood 1993; 81 (03) 734-744
    CrossrefPubMedGoogle Scholar
  • 23 Nguyen DH, Zhou T, Shu J, Mai J-H. Quantifying chromogen intensity in immunohistochemistry via reciprocal intensity. Cancer Incytes 2013; 2 (01) DOI: 10.1038/protex.2013.097.
    CrossrefPubMedGoogle Scholar
  • 24 Gopalakrishnan R, Pendurthi UR, Hedner U, Agersø H, Esmon CT, Rao LV. Influence of endothelial cell protein C receptor on plasma clearance of factor VIIa. J Thromb Haemost 2012; 10 (05) 971-973
    CrossrefPubMedGoogle Scholar
  • 25 Lopez-Fernandez M, Altisent C, Jimenez-Yuste V. , et al. Effective prophylaxis with rFVIIa in young haemophiliacs with inhibitors using a schedule similar to FVIII prophylaxis in non-inhibitor patients. Health 2013; 5: 1151-1157
    PubMedGoogle Scholar
  • 26 Leissinger CA, Singleton T, Kruse-Jarres R. How I use bypassing therapy for prophylaxis in patients with hemophilia A and inhibitors. Blood 2015; 126 (02) 153-159
    PubMedGoogle Scholar
  • 27 Wang M, Lawrence JB, Quon DV. , et al. PERSEPT 1: a phase 3 trial of activated eptacog beta for on-demand treatment of haemophilia inhibitor-related bleeding. Haemophilia 2017; 23 (06) 832-843
    CrossrefPubMedGoogle Scholar
  • 28 Neri Serneri GG, Gensini GF, Gensini RA. Increased capillary permeability in haemophilia and afibrinogenaemia. Acta Haematol 1974; 52 (06) 336-344
    CrossrefPubMedGoogle Scholar
  • 29 O'Doherty MJ, Page CJ, Harrington C, Nunan T, Savidge G. Haemophilia, AIDS and lung epithelial permeability. Eur J Haematol 1990; 44 (04) 252-256
    PubMedGoogle Scholar
  • 30 Sundaram J, Keshava S, Gopalakrishnan R, Esmon CT, Pendurthi UR, Rao LVM. Factor VIIa binding to endothelial cell protein C receptor protects vascular barrier integrity in vivo. J Thromb Haemost 2014; 12 (05) 690-700
    CrossrefPubMedGoogle Scholar
  • 31 Cooke EJ, Zhou JY, Wyseure T. , et al. Vascular permeability and remodelling coincide with inflammatory and reparative processes after joint bleeding in factor VIII-deficient mice. Thromb Haemost 2018; 118 (06) 1036-1047
    Article in Thieme ConnectPubMedGoogle Scholar
  • 32 Valentino LA, Hakobyan N. Histological changes in murine haemophilic synovitis: a quantitative grading system to assess blood-induced synovitis. Haemophilia 2006; 12 (06) 654-662
    CrossrefPubMedGoogle Scholar
  • 33 Ducore J, Alexander WA, Mitchell IS, Lawrence J, Grandoni J, Bonzo D. Potential clinical trial validation of differential EPCR-dependent activity by eptacog beta, a rhFVIIa, in the treatment of bleeding in hemophilia patients with inhibitors. Research and Practice in Thrombosis and Haemostasis 2018; 2 (S1) PB197
    PubMedGoogle Scholar