Internalization and degradation of recombinant human coagulation factor VIIa by the human hepatoma cell line HuH7.
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 approximately 60%, suggesting 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 degrees 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 degrees 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 M(r) approximately 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Chang
G T
GT
Department of Pathology, University of New Mexico School of Medicine, Albuquerque 87131, USA.
Kisiel
W
W
D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D003432:Cross-Linking Reagents; D016895:Culture Media, Serum-Free; D015942:Factor VIIa / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D008113:Liver Neoplasms / Q000378:metabolism*; D009928:Organ Specificity; D011869:Radioligand Assay; D011994:Recombinant Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D003432:Cross-Linking Reagents; D016895:Culture Media, Serum-Free; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D015942:Factor VIIa