Effect of glycoPEGylation on factor VIIa binding and internalization.
Recombinant coagulation factor VIIa (rFVIIa), which is widely used for treatment of bleeding episodes in haemophilia patients with inhibitors, is cleared from the circulation relatively fast with a plasma half-life of 2-4 h. PEGylation is an established and clinically proven strategy for prolonging the circulatory life-time of bio-therapeutic proteins. The aim of this study was to investigate the effect of glycoPEGylation of rFVIIa on rFVIIa binding to its cellular receptors and its subsequent internalization. rFVIIa and glycoPEGylated rFVIIa were labeled with (125)I and the radio-iodinated proteins were used to monitor rFVIIa binding and uptake in endothelial cells and fibroblasts. FVIIa-TF activity at the cell surface was analyzed by a factor X activation assay. Modification of rFVIIa with PEG impaired rFVIIa binding to both endothelial cell protein C receptor and tissue factor (TF) on cell surfaces. The internalization of PEGylated rFVIIa in endothelial cells and fibroblasts was markedly lower compared to the internalization of rFVIIa in these cells. PEGylated rFVIIa was able to activate factor X on TF expressing cell surfaces at a rate similar to that of unmodified rFVIIa when the cells were not subjected to multiple washings to remove the free ligand. General effects such as steric hindrance or changes in electrostatic binding properties of the modified rFVIIa to its receptors are probably responsible for this impairment rather than a loss of specific recognition of the receptors, which could explain near normal activation of factor X by glycoPEGylated rFVIIa on TF expressing cells while its uptake is reduced.
Sen
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P
Center for Biomedical Research, The University of Texas Health Science Center at Tyler, Tyler 75708, USA.
Ghosh
S
S
Ezban
M
M
Pendurthi
U R
UR
Vijaya Mohan Rao
L
L
D001779:Blood Coagulation Factors / Q000502:physiology*; D042783:Endothelial Cells / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism; D015942:Factor VIIa / Q000378:metabolism* / Q000502:physiology; D006031:Glycosylation; D006801:Humans; D011956:Receptors, Cell Surface / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology
D001779:Blood Coagulation Factors; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C533779:glycoPEGylated factor VIIa; D015942:Factor VIIa