Reversible regulation of tissue factor-induced coagulation by glycosyl phosphatidylinositol-anchored tissue factor pathway inhibitor

Endothelial and tumor cells synthesize tissue factor pathway inhibitor (TFP I-1), which regulates tissue factor (TF) function by TF . VIIa . Xa . TFPI- 1 quaternary complex formation (where VIIa and Xa are coagulation factors) and by translocation of these complexes into glycospbingolipid-rich microdo mains of the cell membrane. Recombinant TFPI-1 added exogenously to cells i s targeted to a degradation pathway. This study analyzes whether quaternary complex formation with endogenous TFPI-1 results also in internalization a nd degradation. We demonstrate that endogenous TFPI-1 and recombinant TFPI- 1 differ in their distribution on the cell surface, Recombinant TFPT-1 is f ound in phospholipid- and glycosphingolipid-rich membrane domains, whereas endogenous TFPI-1 preferentially localizes to glycosphingolipid-rich microd omains. On quaternary complex formation, endogenous TFPT-1 remains protease sensitive and accessible for antibodies on intact cells, demonstrating tha t it is not appreciably internalized Rather, regulation of TP by TFPI-1 is restored within 12 hours, consistent with dissociation of quaternary comple xes on the cell surface. Endogenous TFPI-1 can be released from the cell su rface by phospholipase treatment, indicating that TFPI-1 either is a glycos yl phosphatidylinositol (GPI)-anchored protein or binds to a GPI-linked rec eptor. We demonstrate that expression of a recombinant GPI-anchored form of TFPI-1 targets TF . VIIa complexes to glycosphingolipid-rich membrane frac tions. Thus, GPI anchoring of TFPI-1 is sufficient for regulation of TF . V IIa complex function by a pathway of reversible inhibition rather than inte rnalization and degradation.