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 tra nsfected with TF or TF derivatives. Cell surface expression of TF on BHK ce lls was required for VIIa internalization and degradation. Approximately 50 % of cell surface-bound VIIa was internalized in one hour, and a majority o f the internalized VIIa was degraded soon thereafter. Similar rates of VIIa internalization and degradation were obtained with BHK cells transfected w ith a cytoplasmic domain-deleted TF variant or with a substitution of serin e for cysteine at amino acid residue 245 (C245S), Endocytosis of VIIa bound to TF was an active process. Acidification of the cytosol, known to inhibi t the internalization via clathrin-coated pits, did not affect the internal ization of VIIa. Furthermore, receptor-associated protein, known to block b inding of all established ligands to members of the low-density lipoprotein receptor family, was without an effect on the internalization of VIIa. Add ition of tissue factor pathway inhibitor/factor Xa complex did not affect t he internalization rate significantly. A substantial portion (20% to 25%) o f internalized VIIa was recycled back to the cell surface as an intact and functional protein. Although the recycled VIIa constitutes to only approxim ately 10% of available cell surface TF/VIIa sites, it accounts for 65% of t he 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 n ot require the cytoplasmic domain of TF. (Blood, 2001;97:1712-1720) (C) 200 1 by The American Society of Hematology.