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Thromb Haemost 2009; 102(05): 925-935
DOI: 10.1160/TH08-12-0807
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

Factor VIII-eGFP fusion proteins with preserved functional activity for the analysis of the early secretory pathway of factor VIII

Stefan Heinz
1   Institute for Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service Baden-Wuerttemberg-Hesse, Frankfurt/Main, Germany
,
Jörg Schüttrumpf
1   Institute for Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service Baden-Wuerttemberg-Hesse, Frankfurt/Main, Germany
,
Jeremy C. Simpson
2   Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
,
Rainer Pepperkok
2   Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
,
Gerry A. Nicolaes
3   Department of Biochemistry, Maastricht University, Cardiovascular Research Institute Maastricht (CARIM), Maastricht, The Netherlands
,
Daniela Abriss
1   Institute for Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service Baden-Wuerttemberg-Hesse, Frankfurt/Main, Germany
,
Peter Milanov
1   Institute for Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service Baden-Wuerttemberg-Hesse, Frankfurt/Main, Germany
,
Stefanie Roth
1   Institute for Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service Baden-Wuerttemberg-Hesse, Frankfurt/Main, Germany
,
Erhard Seifried
1   Institute for Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service Baden-Wuerttemberg-Hesse, Frankfurt/Main, Germany
,
Torsten Tonn
1   Institute for Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service Baden-Wuerttemberg-Hesse, Frankfurt/Main, Germany
› Author AffiliationsFinancial support: This work was supported by the Hemophilia Bayer Awards Program from Bayer Healthcare Inc. to Stefan Heinz and by the “Stiftung Haemotherapie-Forschung” to Joerg Schuettrumpf. Stefanie Roth and Daniela Abriss are students within the graduate study program “Biologicals, GK-1172” at the Clinic of the Johann Wolfgang Goethe University, funded by the German Research Foundatio
Further Information

Publication History

Received: 14 December 2008

Accepted after major revision: 03 August 2009

Publication Date:
27 November 2017 (online)

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Summary

Considering the difficulty in detecting factor (F)VIII in vivo, fluorescently labelled FVIII protein provides a tool to analyse the intracellular localisation, bio distribution, and pharmacokinetics of the protein in living organisms. Here, we report the use of FVIII full length and B-domain deleted proteins, fused to enhanced green fluorescent protein (eGFP) at the C-terminus of the coagulation protein via a nine amino acid spanning linker. Comparison of the FVIII-eGFP fusion proteins to their unlabelled counterparts showed no impairment with respect to recombinant expression levels, intracellular processing, specific coagulant activity and decay at physiological temperature. Confocal live cell imaging demonstrated ER-Golgi-transport of B-domain deleted FVIII-eGFP in vesicular tubular carriers. Using temperature blocks and release experiments, imaging of FVIII-eGFP fusion proteins enabled for the first time the visualisation of the early secretory pathway of B-domain deleted FVIII in living cells and in particular highlighted the apparent deficit of active transport carriers, an observation consistent with the low rates of FVIII secretion seen in recombinant expression systems.

Keywords

Coagulation - factor VIII - intracellular transport - eGFP - live cell imaging
 

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