title: Tumor necrosis factor-induced endothelial tissue factor is associated with subendothelial matrix vesicles but is not expressed on the apical surface.
abstract: Cultured endothelial cells can be induced by tumor necrosis factor/cachectin (TNF) and other cytokines to synthesize the procoagulant cofactor tissue factor (TF). Intact monolayers of TNF-treated endothelial cells showed only minimal TF activity. In contrast, after permeabilization of these monolayers with detergent (saponin, 0.02%), there was approximately 10- to 20-fold increase in TF-mediated, factor VIIa-dependent factor Xa formation. Extracellular matrix derived from TNF-treated endothelium, prepared after removing the cells by hypotonic lysis or ammonium hydroxide (0.1 N), also had similarly enhanced TF activity. Incubation with a blocking monoclonal antibody to TF inhibited the procoagulant activity of both TNF-stimulated endothelial cells, whether they were intact or permeabilized, and of their matrices. However, when the apical cell surface was pretreated with anti-TF antibody, washed, and then cells were lysed with water or permeabilized with saponin, similar augmentation of TF activity was still observed, suggesting the presence of a pool of TF to which the antibody did not initially gain access. Consistent with this concept, the presence of TF in the matrix of TNF-treated endothelial cells was shown by immunoblotting and morphologic studies; cultured endothelial monolayers and the native endothelium of aortic segments after exposure to TNF showed TF in extracellular matrix, associated with vesicles. In contrast, TF was virtually undetectable on the apical endothelial surface. Taken together, these findings suggest that endothelial TF can be present in a cryptic pool that only gains access to the blood after alteration in the integrity of the endothelial monolayer.
authors.affiliation: Department of Physiology and Cellular Biophysics, Columbia University, College of Physicians and Surgeons, New York, NY 10032.
authors.forename: J
authors.initials: J
authors.lastname: Ryan
authors.forename: J
authors.initials: J
authors.lastname: Brett
authors.forename: P
authors.initials: P
authors.lastname: Tijburg
authors.forename: R R
authors.initials: RR
authors.lastname: Bach
authors.forename: W
authors.initials: W
authors.lastname: Kisiel
authors.forename: D
authors.initials: D
authors.lastname: Stern
mesh_terms: D000906:Antibodies; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000648:ultrastructure; D005109:Extracellular Matrix / Q000737:chemistry / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D008854:Microscopy, Electron; D012503:Saponins / Q000494:pharmacology; D013925:Thromboplastin / Q000032:analysis / Q000096:biosynthesis* / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014471:Umbilical Veins
chemical_list: D000906:Antibodies; D012503:Saponins; D014409:Tumor Necrosis Factor-alpha; D013925:Thromboplastin