title: Localization of tissue factor pathway inhibitor to lipid rafts is not required for inhibition of factor VIIa/tissue factor activity.
abstract: Tissue factor pathway inhibitor (TFPI) abrogates coagulation initiated by the factor VIIa/tissue factor catalytic complex. While the gene structure of TFPI suggests that it is a secreted protein, a large pool of TFPI is associated with the vascular endothelium through its affinity for a glycosylphosphati-dylinositol (GPI)-linked membrane protein. Inhibition of tissue factor by TFPI coincides with the translocation of quaternary complexes containing tissue factor, factor VIIa, factor Xa, and TFPI to detergent-insoluble plasma membrane domains rich in cholesterol, sphingomyelin, and GPI-linked proteins known as lipid rafts and caveolae. It is not known if localization of TFPI to these membrane domains is required for its inhibition of tissue factor procoagulant activity. We generated chimeric TFPI molecules linked directly to the plasma membrane via a GPI anchor or hydrophobic transmembrane domain and expressed these in HEK293 cells that produce tissue factor but not endogenous TFPI. The GPI-anchored chimera was exclusively enriched in detergent-insoluble membrane fractions while the transmembrane molecule was not. Transfectants expressing equal levels of the GPI-linked or transmembrane TFPI displayed equal anticoagulant potency as assessed by tissue factor-mediated conversion of factor X to factor Xa. Disruption of lipid rafts with cyclodextrin likewise had no effect on the inhibitory activity of the transmembrane or GPI-linked TFPI chimeras in HEK293 cells, nor on endogenous TFPI expressed by ECV304 cells. Thus, we conclude that the GPI anchor and membrane localization to lipid rafts does not enhance inhibition of factor VIIa/tissue factor by cell-surface associated TFPI.
authors.affiliation: Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110, USA. Dietzen_D@kids.wustl.edu
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Dietzen
authors.forename: Garnet G
authors.initials: GG
authors.lastname: Jack
authors.forename: Keith L
authors.initials: KL
authors.lastname: Page
authors.forename: Tina A
authors.initials: TA
authors.lastname: Tetzloff
authors.forename: Connie L
authors.initials: CL
authors.lastname: Hall
authors.forename: Alan E
authors.initials: AE
authors.lastname: Mast
mesh_terms: D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003505:Cyclodextrins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015942:Factor VIIa / Q000037:antagonists & inhibitors*; D017261:Glycosylphosphatidylinositols / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D021962:Membrane Microdomains / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013925:Thromboplastin / Q000037:antagonists & inhibitors*; D014162:Transfection
chemical_list: D003505:Cyclodextrins; D017261:Glycosylphosphatidylinositols; D008074:Lipoproteins; D011993:Recombinant Fusion Proteins; C051928:lipoprotein-associated coagulation inhibitor; D013925:Thromboplastin; D015942:Factor VIIa