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

Stefan Heinz; Jörg Schüttrumpf; Jeremy C. Simpson; Rainer Pepperkok; Gerry A. Nicolaes; Daniela Abriss; Peter Milanov; Stefanie Roth; Erhard Seifried; Torsten Tonn

doi:10.1160/TH08-12-0807

Thrombosis and Haemostasis, Inhaltsverzeichnis

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

¹Institute for Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service Baden-Wuerttemberg-Hesse, Frankfurt/Main, Germany

,

Jörg Schüttrumpf

¹Institute for Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service Baden-Wuerttemberg-Hesse, Frankfurt/Main, Germany

,

Jeremy C. Simpson

²Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

,

Rainer Pepperkok

²Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

,

Gerry A. Nicolaes

³Department of Biochemistry, Maastricht University, Cardiovascular Research Institute Maastricht (CARIM), Maastricht, The Netherlands

,

Daniela Abriss

¹Institute for Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service Baden-Wuerttemberg-Hesse, Frankfurt/Main, Germany

,

Peter Milanov

¹Institute for Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service Baden-Wuerttemberg-Hesse, Frankfurt/Main, Germany

,

Stefanie Roth

¹Institute for Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service Baden-Wuerttemberg-Hesse, Frankfurt/Main, Germany

,

Erhard Seifried

¹Institute for Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service Baden-Wuerttemberg-Hesse, Frankfurt/Main, Germany

,

Torsten Tonn

¹Institute for Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service Baden-Wuerttemberg-Hesse, Frankfurt/Main, Germany

› Institutsangaben

Abstract

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

Volltext

Referenzen

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