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Thromb Haemost 2010; 104(06): 1166-1173
DOI: 10.1160/TH10-06-0356
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

The unsialylated subpopulation of recombinant activated factor VII binds to the asialo-glycoprotein receptor (ASGPR) on primary rat hepatocytes

Torben Seested
1   Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Copenhagen, Denmark
2   Exploratory ADME, Biopharmaceutical Research Unit, Novo Nordisk A/S, Måløv, Denmark
,
Hanne M. Nielsen
1   Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Copenhagen, Denmark
,
Erik I. Christensen
3   Department of Anatomy, Section of Cell Biology, Aarhus University, Aarhus C, Denmark
,
Rupa S. Appa
2   Exploratory ADME, Biopharmaceutical Research Unit, Novo Nordisk A/S, Måløv, Denmark
› Author Affiliations Financial support: For financial support, we wish to thank the Drug Research Academy at the Faculty of Pharmaceutical Sciences, University of Copenhagen, Denmark.
Further Information

Publication History

Received: 08 June 2010

Accepted after major revision: 24 August 2010

Publication Date:
24 November 2017 (online)

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Summary

Recombinant activated factor VII (rFVIIa; NovoSeven®) is a heterogeneously glycosylated serine protease used for treatment of haemophiliacs with inhibitors. The drug substance contains a subpopulation consisting of ~20% of rFVIIa molecules which are unsialylated and consists of carbohydrate moieties with terminally exposed galactose and N-acetyl-D-galactosamine (GalNAc). Recently, data from an in situ per-fused liver model showed that a subpopulation of rFVIIa, appearing to be unsialylated rFVIIa, was cleared by the liver, thus suggesting a carbohydrate-moiety mediated mechanism. The parenchymal cells of the liver, hepatocytes, are known to abundantly express functional carbohydrate-specific receptors and in this study we therefore used primary rat hepatocytes to study binding and intracellular fate of rFVIIa at a cellular level. Immunofluorescence microscopy showed that rFVIIa was distributed into distinct intracellular vesicles and electron microscopic autoradiography revealed that radioiodinated rFVIIa distributed only into cytoplasmic free vesicles resembling endosomes and lysosomes. These findings suggest that endocytosis of rFVIIa in hepatocytes could be partly mediated via initial membrane binding to a receptor. Quantitative binding studies showed that the presence of excess unlabelled asialo-orosomucoid, asialo-rFVIIa and GalNAc significantly decreased binding of 125I-rFVIIa. An antibody which specifically binds to the carbohydrate recognition domain of the asialoglycoprotein receptor (ASGPR) significantly decreased binding of asialo-rFVIIa by ~36% and rFVIIa by ~19%. Together our data showed that a receptor-mediated mechanism involving the ASGPR is able to bind a subpopulation of unsialylated rFVIIa, while a hepatic mechanism for binding and clearing sialylated rFVIIa is still unknown.

Keywords

Recombinant activated factor VII (rFVIIa) - primary rat hepatocytes - electron microscopic autoradiography - asialoglycoprotein receptor (ASGPR) - asialo-orosomucoid (ASOR)
 

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