Studies on rabbit natural and recombinant tissue factors : intracellular retention and regulation of surface expression in cultured cells

• Published in: American journal of physiology. Heart and circulatory physiology Vol. 57; no. 5; pp. H2192 - H2202
• Main Authors: FORTIN, Jean-Philippe, RIVARD, Georges E, ADAM, Albert, MARCEAU, Francois
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Physiological Society 01.05.2005
• Subjects: Biological and medical sciences; Cell culture; Fundamental and applied biological sciences. Psychology; Gene expression; Genetic recombination; Rabbits; Tissues; Vertebrates: cardiovascular system; lipid rafts; kinin B1 receptors; Tissue factor; Kinin; Rabbit; Lipids; Smooth muscle; microparticles; Lagomorpha; In vitro; Cell surface; smooth muscle cells; Vertebrata; Mammalia; Circulatory system; Intracellular; Biological receptor
• ISSN: 0363-6135; 1522-1539

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 about 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. [PUBLICATION ABSTRACT]