INTERNALIZATION AND DEGRADATION OF RECOMBINANT HUMAN COAGULATION-FACTOR VIIA BY THE HUMAN HEPATOMA-CELL LINE HUH7

Previous studies demonstrated that several normal and transformed cult ured human cell lines specifically bind human coagulation factors VII and VIIa via tissue factor. In the present study, we show that I-125-r adiolabeled recombinant human factor VIIa (I-125-rFVIIa) binds to a hu man hepatoma cell line (HuH7). In the presence of rabbit polyclonal an ti-human tissue factor apoprotein IgG, binding of I-125-rFVIIa to the HuH7 cells was decreased similar to 60%, suggesting the presence of ti ssue factor-independent binding sites for I-125-rFVIIa on these cells. The binding isotherm of I-125-rFVIIa for the HuH7 cells in the presen ce of anti-tissue factor IgG exhibited a hyperbolic profile and was ti me-, temperature-and calcium-dependent. Furthermore, binding at 4 degr ees C was specific, dose-dependent and saturable. Scatchard analysis o f 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 cel l. At 4 degrees C, I-125-rFVIIa bound to, and eluted from, the cell wa s indistinguishable from offered I-125-rFVIIa as judged by sodium dode cyl sulfate-polyacrylamide gel electrophoresis followed by autoradiogr aphy. The molecular properties of the tissue factor-independent bindin g protein were studied by using the cleavable cross-linking agent 3,3' -dithiobis(sulfosuccinimidylpropionate ate). A cross-linking product o f I-125-rFVIIa and a cell surface protein with an apparent Mr similar to 100,000 was observed. The cross-linking reaction was strongly inhib ited by a 100-fold molar excess of unlabeled rFVIIa, but not by rabbit polyclonal anti-human tissue factor apoprotein IgG, indicating that c ross-linking does not involve the extracellular domain of tissue facto r. After binding, internalization of I-125-rFVIla by the HuH7 cells wa s observed al 37 degrees C in the presence of rabbit polyclonal anti-h uman tissue factor apoprotein IgG. Internalization of I-125-rFVIIa pro ceeds at a rate of 0.4 fmol I-125-rFVIIa/min/l0(6) cells, reaching a s teady state after 2 h. Following binding and internalization, I-125-rF VIIa reappeared in the culture medium within 30 min, at approximately the same rate as internalized I-125-rFVIIa. This I-125-rFVIIa probably represents degradation of I-125-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 I-125-rFVIIa, suggesting that degr adation presumably occurs via a lysosomal-dependent pathway. These stu dies demonstrate that the liver may play an important role in the clea rance mechanism(s) of FVIIa.