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

¹Centre de Recherche, Centre Hospitalier Universitaire de Québec, Québec; ²Division of Hematology, Hôpital Sainte Justine, Montreal; and ³Faculté de Pharmacie, Université de Montréal, Montreal, Québec, Canada

Submitted 10 November 2004 ; accepted in final form 10 January 2005

Tissue factor (TF) is the most important trigger of blood coagulation in vascular pathology. Rabbit TF, with or without (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 TFC-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, TFC-GFP was 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 TFC-GFP. In serum-starved SMCs, the expression of endogenous TF was upregulated by interleukin-1 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.

smooth muscle cells; microparticles; kinin B₁ receptors; lipid rafts

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