title: Studies on rabbit natural and recombinant tissue factors: intracellular retention and regulation of surface expression in cultured cells.
abstract: Tissue factor (TF) is the most important trigger of blood coagulation in vascular pathology. Rabbit TF, with or without (delta C) its COOH-terminal intracellular tail, has been conjugated to green fluorescent protein (GFP) to study subcellular localization and other functions of TF. TF-GFP and TF delta C-GFP are associated with Na2CO3-resistant buoyant fractions in HEK-293 cells (lipid rafts); there is no morphological difference in the surface distribution of these or other GFP-labeled membrane proteins present in or excluded from rafts (confocal microscopy, HEK-293 cells). Endogenous TF expressed by rabbit aortic smooth muscle cells (SMCs) is also raft associated. Membranes from HEK-293 cells expressing recombinant TF-GFP or wild-type TF were equipotent to clot human plasma; however, TF delta C-GFP was approximately 20-fold more active (per membrane weight). Immunoblot confirmed that the deletion mutant is more abundantly expressed, and confocal microscopy showed that it has preferential membrane localization, whereas TF-GFP is mainly intracellular (nuclear lining and multiple granules). With a similar half-life (<4 h), the two constructions differ by their intracellular retention, lower for TF delta C-GFP. In serum-starved SMCs, the expression of endogenous TF was upregulated by interleukin-1 beta and/or FBS treatment (immunoblot, immunofluorescence, clotting assay). However, TF secretion or surface expression was not regulated by stimuli of physiological intensity (such as stimulation of the coexpressed kinin B1 receptors), although a calcium ionophore was highly active in this respect. TF is a raft-associated molecule whose surface expression (secretion) is apparently retarded or impaired by structural determinant(s) located in its COOH-terminal tail.
authors.affiliation: Centre de Recherche, Centre Hospitalier Universitaire de Québec, Québec, Canada.
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Fortin
authors.forename: Georges E
authors.initials: GE
authors.lastname: Rivard
authors.forename: Albert
authors.initials: A
authors.lastname: Adam
authors.forename: François
authors.initials: F
authors.lastname: Marceau
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology; D001483:Base Sequence; D002478:Cells, Cultured; D015870:Gene Expression / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D007668:Kidney / Q000166:cytology; D021962:Membrane Microdomains / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000502:physiology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013925:Thromboplastin / Q000235:genetics / Q000378:metabolism*
chemical_list: D007476:Ionophores; D008565:Membrane Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D013925:Thromboplastin