Tissue factor-mediated endocytosis, recycling, and degradation of factor VIIa by a clathrin-independent mechanism not requiring the cytoplasmic domain of tissue factor.
Endocytosis and recycling of coagulation factor VIIa (VIIa) bound to tissue factor (TF) was investigated in baby hamster kidney (BHK) cells stably transfected with TF or TF derivatives. Cell surface expression of TF on BHK cells was required for VIIa internalization and degradation. Approximately 50% of cell surface-bound VIIa was internalized in one hour, and a majority of the internalized VIIa was degraded soon thereafter. Similar rates of VIIa internalization and degradation were obtained with BHK cells transfected with a cytoplasmic domain-deleted TF variant or with a substitution of serine for cysteine at amino acid residue 245 (C245S). Endocytosis of VIIa bound to TF was an active process. Acidification of the cytosol, known to inhibit the internalization via clathrin-coated pits, did not affect the internalization of VIIa. Furthermore, receptor-associated protein, known to block binding of all established ligands to members of the low-density lipoprotein receptor family, was without an effect on the internalization of VIIa. Addition of tissue factor pathway inhibitor/factor Xa complex did not affect the internalization rate significantly. A substantial portion (20% to 25%) of internalized VIIa was recycled back to the cell surface as an intact and functional protein. Although the recycled VIIa constitutes to only approximately 10% of available cell surface TF/VIIa sites, it accounts for 65% of the maximal activation of factor X by the cell surface TF/VIIa. In summary, the present data provide evidence that TF-dependent internalization of VIIa in kidney cells occurs through a clathrin-independent mechanism and does not require the cytoplasmic domain of TF.
Department of Tissue Factor/Factor VIIa (TF/VIIa) Research, Health Care Discovery, Novo Nordisk A/S, Maalov, Denmark.
C B
CB
Hansen
C
C
Pyke
L C
LC
Petersen
L V
LV
Rao
D000818:Animals; D002460:Cell Line; D002966:Clathrin; D006224:Cricetinae; D004705:Endocytosis / Q000187:drug effects* / Q000502:physiology; D015942:Factor VIIa / Q000187:drug effects / Q000493:pharmacokinetics / Q000502:physiology*; D015951:Factor Xa / Q000494:pharmacology; D007457:Iodine Radioisotopes; D008074:Lipoproteins / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D013925:Thromboplastin / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D014162:Transfection
D002966:Clathrin; D007457:Iodine Radioisotopes; D008074:Lipoproteins; C051928:lipoprotein-associated coagulation inhibitor; D013925:Thromboplastin; D015942:Factor VIIa; D015951:Factor Xa