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)