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Thromb Haemost 1995; 73(02): 231-238
DOI: 10.1055/s-0038-1653756
Original Article
Coagulation
Schattauer GmbH Stuttgart

Internalization and Degradation of Recombinant Human Coagulation Factor VIIa by the Human Hepatoma Cell Line HuH7

Glenn T G Chang
The Department of Pathology, University of New Mexico School of Medicine, Albuquerque, NM, USA
,
Walter Kisiel
The Department of Pathology, University of New Mexico School of Medicine, Albuquerque, NM, USA
› Author Affiliations
Further Information

Publication History

Received 08 June 1994

Accepted after resubmission 18 October 1994

Publication Date:
09 July 2018 (online)

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Summary

Previous studies demonstrated that several normal and transformed cultured human cell lines specifically bind human coagulation factors VII and VIIa via tissue factor. In the present study, we show that 125I- radiolabeled recombinant human factor VIIa (125I-rFVIIa) binds to a human hepatoma cell line (HuH7). In the presence of rabbit polyclonal anti-human tissue factor apoprotein IgG, binding of 125I-rFVIIa to the HuH7 cells was decreased ∼60%, suggesting the presence of tissue factor-independent binding sites for 125I-rFVIIa on these cells. The binding isotherm of 125I-rFVIIa for the HuH7 cells in the presence of anti-tissue factor IgG exhibited a hyperbolic profile and was time-, temperature- and calcium-dependent. Furthermore, binding at 4° C was specific, dose-dependent and saturable. Scatchard analysis of the binding data demonstrated a single class of binding sites with a dissociation constant (Kd) of 3.2 nM and 27,000 binding sites per cell. At 4° C, 125I-rFVIIa bound to, and eluted from, the cell was indistinguishable from offered 125I-rFVIIa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography.

The molecular properties of the tissue factor-independent binding protein were studied by using the cleavable cross-linking agent 3, 3’- dithiobis(sulfosuccinimidylpropionate). A cross-linking product of 125I-rFVIIa and a cell surface protein with an apparent Mr ∼100,000 was observed. The cross-linking reaction was strongly inhibited by a 100-fold molar excess of unlabeled rFVIIa, but not by rabbit polyclonal anti-human tissue factor apoprotein IgG, indicating that cross-linking does not involve the extracellular domain of tissue factor.

After binding, internalization of 125I-rFVIIa by the HuH7 cells was observed at 37° C in the presence of rabbit polyclonal anti-human tissue factor apoprotein IgG. Internalization of 125I-rFVIIa proceeds at a rate of 0.4 fmol 125I-rFVIIa/min/106 cells, reaching a steady state after 2 h. Following binding and internalization, 125I-rFVIIa reappeared in the culture medium within 30 min, at approximately the same rate as internalized 125I-rFVIIa. This 125I-rFVIIa probably represents degradation of 125I-rFVIIa into small peptides, since it could not be precipitated by rabbit polyclonal affinity-purified anti-FVII IgG or by trichloroacetic acid. Chloroquine treatment of the HuH7 cells inhibited the degradation of 125I-rFVIIa, suggesting that degradation presumably occurs via a lysosomal-dependent pathway. These studies demonstrate that the liver may play an important role in the clearance mechanism(s) of FVIIa.

 

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