title: Activation of the coagulation mechanism on tumor necrosis factor-stimulated cultured endothelial cells and their extracellular matrix. The role of flow and factor IX/IXa.
abstract: Infusion of tumor necrosis factor (TNF) into tumor-bearing mice led to intravascular clot formation with fibrin deposition in microvessels in the tumor bed in close association with the vessel wall, which could be prevented by active site-blocked factor IXa (IXai). This observation prompted us to examine the role of the intrinsic system in activation of the coagulation mechanism on TNF-stimulated human endothelial cell monolayers and endothelial-derived matrix during exposure to purified coagulation factors or flowing blood. Treatment of endothelial cells in intact monolayers with TNF induced expression of the procoagulant cofactor tissue factor (TF) in a dose-dependent manner, and after removal of the cells, TF was present in the matrix. TNF-treated endothelial cell monolayers exposed to blood anticoagulated with low molecular weight heparin induced activation of coagulation. Addition of IXai blocked the procoagulant response on TNF-treated endothelial cells, and consistent with this, the presence of factor IX/VIIIa enhanced endothelial TF/factor VII(a) factor X activation over a wide range of cytokine concentrations (0-600 pM). When TF-dependent factor X activation on endothelial cells was compared with preparations of subendothelium, the extracellular matrix was 10-20 times more effective. IXai blocked TF/factor VII(a) mediated activated coagulation on matrix, but only at lower concentration of TNF (less than 50 pM). Similarly, enhancement of factor Xa formation on matrix by factors IX/VIIIa was most evident at lower TNF concentrations. When anticoagulated whole blood flowing with a shear of 300 s-1 was exposed to matrices from TNF-treated endothelial cells, but not matrices from control cells, fibrinopeptide A (FPA) generation, fibrin deposition, and platelet aggregate formation were observed. FPA generation could be prevented by a blocking antibody to TF and by active site-blocked factor Xa (Xai) over a wide range of TNF concentrations (0-600 pM), whereas IXai only blocked FPA generation at lower TNF concentrations (less than 50 pM). Activation of coagulation on matrix from TNF-stimulated endothelial cells was dependent on the presence of platelets, indicating the important role of platelets in propagating the reactions leading to fibrin formation. These observations demonstrate the potential of cytokine-stimulated endothelium and their matrix to activate coagulation and suggest the importance of the intrinsic system in factor Xa formation on cellular surfaces.
authors.affiliation: Department of Hematology, University Hospital, Utrecht, The Netherlands.
authors.forename: P N
authors.initials: PN
authors.lastname: Tijburg
authors.forename: J
authors.initials: J
authors.lastname: Ryan
authors.forename: D M
authors.initials: DM
authors.lastname: Stern
authors.forename: B
authors.initials: B
authors.lastname: Wollitzky
authors.forename: S
authors.initials: S
authors.lastname: Rimon
authors.forename: A
authors.initials: A
authors.lastname: Rimon
authors.forename: D
authors.initials: D
authors.lastname: Handley
authors.forename: P
authors.initials: P
authors.lastname: Nawroth
authors.forename: J J
authors.initials: JJ
authors.lastname: Sixma
authors.forename: P G
authors.initials: PG
authors.lastname: de Groot
mesh_terms: D000818:Animals; D001777:Blood Coagulation; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects*; D005109:Extracellular Matrix / Q000187:drug effects*; D005164:Factor IX / Q000502:physiology*; D015949:Factor IXa / Q000502:physiology*; D005170:Factor X / Q000378:metabolism; D005337:Fibrin / Q000378:metabolism; D005354:Fibrosarcoma / Q000473:pathology; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D010974:Platelet Aggregation / Q000187:drug effects; D011863:Radioimmunoassay; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D014409:Tumor Necrosis Factor-alpha; D005164:Factor IX; D005170:Factor X; D005337:Fibrin; D015949:Factor IXa