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

Torben Seested Johansen; Hanne Mørck Nielsen; Erik Ilsø Christensen; Rupa S. Appa

doi:10.1160/th10-06-0356

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

Torben Seested Johansen, Hanne Mørck Nielsen, Erik Ilsø Christensen, Rupa S. Appa

13 Citations (Scopus)

Abstract

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 perfused 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.

Original language	English
Journal	Thrombosis and Haemostasis
Volume	104
Issue number	6
Pages (from-to)	1166-1173
ISSN	0340-6245
DOIs	https://doi.org/10.1160/th10-06-0356
Publication status	Published - Dec 2010

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Former Faculty of Pharmaceutical Sciences

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10.1160/th10-06-0356

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The unsialylated subpopulation of recombinant activated factor VII binds to the asialo-glycoprotein receptor (ASGPR) on primary rat hepatocytes. / Johansen, Torben Seested; Nielsen, Hanne Mørck; Christensen, Erik Ilsø et al.

In: Thrombosis and Haemostasis, Vol. 104, No. 6, 12.2010, p. 1166-1173.

Research output: Contribution to journal › Journal article › Research › peer-review

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

abstract = "Recombinant activated factor VII (rFVIIa; NovoSeven{\textregistered}) 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 perfused 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.",

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author = "Johansen, {Torben Seested} and Nielsen, {Hanne M{\o}rck} and Christensen, {Erik Ils{\o}} and Appa, {Rupa S.}",

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doi = "10.1160/th10-06-0356",

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T1 - The unsialylated subpopulation of recombinant activated factor VII binds to the asialo-glycoprotein receptor (ASGPR) on primary rat hepatocytes

AU - Johansen, Torben Seested

AU - Nielsen, Hanne Mørck

AU - Christensen, Erik Ilsø

AU - Appa, Rupa S.

PY - 2010/12

Y1 - 2010/12

N2 - 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 perfused 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.

AB - 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 perfused 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.

KW - Former Faculty of Pharmaceutical Sciences

U2 - 10.1160/th10-06-0356

DO - 10.1160/th10-06-0356

M3 - Journal article

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SN - 0340-6245

VL - 104

SP - 1166

EP - 1173

JO - Thrombosis et diathesis haemorrhagica

JF - Thrombosis et diathesis haemorrhagica

IS - 6

ER -

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

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