Reversible regulation of tissue factor-induced coagulation by glycosyl phosphatidylinositol-anchored tissue factor pathway inhibitor.
Endothelial and tumor cells synthesize tissue factor pathway inhibitor (TFPI-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 glycosphingolipid-rich microdomains of the cell membrane. Recombinant TFPI-1 added exogenously to cells is targeted to a degradation pathway. This study analyzes whether quaternary complex formation with endogenous TFPI-1 results also in internalization and degradation. We demonstrate that endogenous TFPI-1 and recombinant TFPI-1 differ in their distribution on the cell surface. Recombinant TFPI-1 is found in phospholipid- and glycosphingolipid-rich membrane domains, whereas endogenous TFPI-1 preferentially localizes to glycosphingolipid-rich microdomains. On quaternary complex formation, endogenous TFPI-1 remains protease sensitive and accessible for antibodies on intact cells, demonstrating that it is not appreciably internalized. Rather, regulation of TF by TFPI-1 is restored within 12 hours, consistent with dissociation of quaternary complexes on the cell surface. Endogenous TFPI-1 can be released from the cell surface by phospholipase treatment, indicating that TFPI-1 either is a glycosyl phosphatidylinositol (GPI)-anchored protein or binds to a GPI-linked receptor. We demonstrate that expression of a recombinant GPI-anchored form of TFPI-1 targets TF. VIIa complexes to glycosphingolipid-rich membrane fractions. Thus, GPI anchoring of TFPI-1 is sufficient for regulation of TF. VIIa complex function by a pathway of reversible inhibition rather than internalization and degradation.
Ott
I
I
Deutsches Herzzentrum, Munich, Germany.
Miyagi
Y
Y
Miyazaki
K
K
Heeb
M J
MJ
Mueller
B M
BM
Rao
L V
LV
Ruf
W
W
D000818:Animals; D000990:Antithrombin III / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites / Q000502:physiology; D001777:Blood Coagulation / Q000502:physiology*; D016466:CHO Cells; D006224:Cricetinae; D003902:Detergents; D015536:Down-Regulation / Q000502:physiology; D015942:Factor VIIa / Q000378:metabolism; D005170:Factor X / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D010455:Peptides; D010940:Plant Proteins; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D012995:Solubility; D013925:Thromboplastin / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000378:metabolism
D003902:Detergents; D017261:Glycosylphosphatidylinositols; C053865:Kunitz-type protease inhibitor, plant; D008565:Membrane Proteins; D010455:Peptides; D010940:Plant Proteins; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; D000990:Antithrombin III; D005170:Factor X; D013925:Thromboplastin; D015942:Factor VIIa; D015951:Factor Xa