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

Journal: Thrombosis and Haemostasis
ISSN: 0340-6245
Topic:

Heparin and its derivatives – Present and future

DOI: http://dx.doi.org/10.1160/TH08-12-0807
Issue: 2009: 102/5 (Nov) pp. 799-1006
Pages: 925-935

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

Stefan Heinz1; Jörg Schüttrumpf1; Jeremy C. Simpson2; Rainer Pepperkok2; Gerry A. Nicolaes3; Daniela Abriss1; Peter Milanov1; Stefanie Roth1; Erhard Seifried1; Torsten Tonn1

1Institute for Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service Baden-Wuerttemberg-Hesse, Frankfurt/Main, Germany; 2Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany; 3Department of Biochemistry, Maastricht University, Cardiovascular Research Institute Maastricht (CARIM), Maastricht, The Netherlands

Keywords

coagulation, factor VIII, intracellular transport, eGFP, live cell imaging

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.

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