Previous studies demonstrated that endothelial cells participate actively in both anticoagulant and procoagulant reactions. Although anticoagulant mechanisms predominate on the surface of quiescent endothelial cells, perturbed endothelial cells can promote coagulation through the coordinated induction of procoagulant activity and suppression of anticoagulant mechanisms. Purified recombinant interleukin 1 was infused intravenously into rabbits and coagulant 'properties of the native aortic endothelium were subsequently studied. Interleukin 1 infusion resulted in a timeand dose-dependent induction of the procoagulant cofactor tissue factor, while concomitantly blocking~the protein C anticoagulant pathway. Tissue factor activity increased >10-fold by 3-5 hr after the infusion,. while endothelial celldependent thrombin-mediated protein; C activation decreased by 72% and assembly of functional activated protein C-protein S complex, on the vessel surface was decreased by >90%. This has led us to examine in vivo the effect of IL-1 infusion in the rabbit on procoagulant and anticoagulant properties of the native aortic endothelium. MATERIALS AND METHODSReagents and Assays. Infusion studies were carried out using recombinant murine IL-1 (15), generously provided by Peter LoMedico (Hoffmann La Roche). Heat treatment of IL-1 was carried out at 90'C for 70 min (19) and resulted in loss of IL-1 activity in the thymocyte costimulator assay (20). Control protein, a single-chain polypeptide ofMr 17,000, was also purified from Escherichia coli (provided by Peter LoMedico). This protein, distinct from IL-1, has no IL-1 or other known biologic activity and was also free of detectable endotoxin.Human factor X (130 units/ml) was purified to homogeneity by the method of DiScipio et al. (21), and purified human factor VIIa (22) was generously provided by W. Kisiel (University of New Mexico, Albuquerque, NM). These reagents Were used to assay the tissue factor by incubating samples with factors VIIa (8 nM) and X (1.1 aM) for 10 min at 21'C in incubation buffer [10 mM Hepes (pH 7.45) containing 137 mM NaCl, 4 mM KCl, 11 mM glucose, 2.5 mM CaCl2, and fatty acid-free bovine serum albumin (2 mg/ml)] at a final vol of 0.1 ml. One sample was removed from each reaction mixture, added to 0.5 ml of 50 mM Tris HCl, pH 7.8/175 mM NaCl/10 mM EDTA/ovalbumin (0.5 mg/ml), and assayed for factor Xa by monitoring hydrolysis at 405 nM of the chromogenic substrate S2222 (Bz-Ile-Glu-Gly-Arg-pnitroanilide; Helena Laboratories, Beaumont; TX) (0.1 ml; 0.5 nM) (23). Factors VIta and X were present at saturating concentrations and factor Xa formation, which was linear, was limited only by the amount of tissue factor. a-Thrombin (2.5 units/,ug), antithrombin III (inhibitory activity of 1.0 unit of thrombin per pug), and protein C were purified to homogeneity from bovine plasma, as described (24-26). Thrombin-mediated protein C activation was studied as described (18) Human protein S and bovine factor Va were purified to homogeneity as described (21, 28)...

Tumor necrosis factor/cachectin (TNF) has been implicated as a mediator of the host response in sepsis and neoplasia. Recent work has shown that TNF can modulate endothelial cell hemostatic properties, suggesting that endothelium is a target tissue for TNF. This led us to examine whether endothelial cells have specific binding sites for TNF and augment the biological response to TNF by elaborating the inflammatory mediator, IL-1. Incubation of 125I-recombinant human TNF with confluent, cultured human umbilical vein endothelial cells resulted in time-dependent, reversible, and saturable binding. Binding was half-maximal at a TNF concentration of 105 +/- 40 pM, and at saturation 1,500 molecules were bound per cell. Heat-treated TNF, which is biologically inactive, did not bind to endothelium. In addition to surface binding, TNF induced the elaboration of IL-1 activity by endothelial cells in a time-dependent manner. Generation of IL-1 activity required protein synthesis and was half-maximal at a TNF concentration of 50 +/- 20 pM. IL-1 activity from TNF-treated endothelium could be adsorbed by an immobilized antibody to IL-1. Heat-treated TNF was ineffective in eliciting endothelial cell IL-1. These data indicate that TNF can bind specifically to endothelium and initiate a cascade of inflammatory and coagulant events on the vessel surface potentially central to the host response to neoplasia and sepsis.

Bovine aortic smooth muscle cells in vitro responded to 1 nM to 10 ,M serotonin with increased incorporation of [3H]thymidine into DNA. The mitogenic effect of serotonin was half-maximal at 80 nM and maximal above 1 ,uM. At a concentration of 1 ,uM, serotonin stimulated smooth muscle cell mitogenesis to the same extent as human plateletderived growth factor (PDGF) at 12 ng/ml, Tryptamine was =1/10th as potent as serotonin as a mitogen for smooth muscle cells. Other indoles that are structurally related to serotonin (Dand L-tryptophan, 5-hydroxy-L-tryptophan, N-acetyl-5-hydroxytryptamine, melatonin, 5-hydroxyindoleacetic acid, and 5-hydroxytryptophol) and quipazine were inactive. The stimulatory effect of serotonin on smooth muscle cell DNA synthesis required prolonged (20-24 hr) [3HlThymidine Incorporation. Bovine aortic smooth muscle cells from frozen stock (passages 3-12) were suspended in Dulbecco's modified Eagle's medium (DMEM) (pH 7.4) supplemented with 10% calf serum/6 mM glutamine/ penicillin (100 units/ml)/streptomycin (100 pug/ml) for seeding into 16-mm 96-well microtiter plates at a density of w50,000 cells per well. After 48 hr, the growth medium was replaced with serum-free quiescing medium consisting of DMEM and Ham's F12 (1:1; pH 7.4) supplemented with ovalbumin (1 mg/ml)/ascorbic acid (25 ,ug/ml)/selenium (25 ng/ml)/dialyzed Cohn fraction IV (10 ug/ml)/epidermal growth factor (10 ng/ml)/transferrin 10 ,tg/ml) 50 ,uM hydrocortisone/6 mM glutamine/penicillin (100 units/ml)/ streptomycin (100 ,ug/ml) (8). Smooth muscle cells were incubated in the quiescing medium for 96 hr. The medium was changed once during this period. Compounds to be tested were added to smooth muscle cells in quiescing medium 1 hr before serotonin and/or PDGF. The cultures were exposed to serotonin and/or PDGF for 20 hr before [3H]thymidine (2.0 ,uCi/ml; 1 Ci = 37 GBq) was included in the quiescing medium. Four hours after the addition of [3H]thymidine, experiments were terminated by aspirating the medium and subjecting the cultures to sequential washes with Dulbecco's phosphate-buffered saline (pH 7.4) containing 1 mM CaCl2, 1 mM MgCl2, 10% trichloroacetic acid, and ethanol/ether (2:1). Phase-contrast microscopy was used to inspect the microwells for evidence of cell detachment or changes in cell morphology. Acid-insoluble [3H]thymidine was extracted into 0.5 M NaOH and quantified by using a liquid scintillation counter.Materials. Serotonin, tryptamine, L-tryptophan, Dtryptophan, N-acetyl-5-hydroxytryptamine, 5-hydroxy-Ltryptophan, melatonin, 5-hydroxyindoleacetic acid, 5-hydroxytryptophol, propranolol, atropine, cimetidine, 1-methyl-3-isobutylxanthine, ovalbumin, and Cohn fraction IV were obtained from Sigma; quipazine (2-[1-piperazi- 674The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Although the endothelial cell is considered antithrombogenic, endothelium has recently been shown to participate in procoagulant reactions. In this report cultured bovine aortic endothelial cells are shown to propagate a procoagulant pathway starting with factor XIa, leading to activation of factors IX, VIII, X, and prothrombin, culminating in fibrinopeptide A cleavage from fibrinogen and formation of a fibrin clot. Electron microscopic studies demonstrated that fibrin strands are closely associated with the endothelial cells. Endotoxin-treated endothelial cells, having acquired tissue factor activity, generated fibrinopeptide A in the presence of factors VII,, IX, VIII, X, prothrombin, and fibrinogen. Factor X activation by factor VIII and tissue factor expressed by endothelial cells is 10 times greater in the presence of factors IX and VIII than in their absence. This indicates that on the perturbed endothelial cell surface, factors IX and VIII do have an important role in the activation of factor X. Addition of platelets (108 per ml) augmented thrombin formation seen in the presence of endothelium alone by about 15-fold. Anti-human factor V IgG decreased this enhanced thrombin formation in the presence of platelets, indicating that factor V from platelets was playing an important role in thrombin formation. These data lead us to propose that endothelial cells can actively participate in procoagulant reactions. Although platelets can augment thrombin formation by these endothelial celldependent reactions, endothelial cells alone can lead to formation of a cell-associated fibrin clot. The endotoxin-treated endothelial cell provides a model of the thrombotic state supplying tissue factor to initiate coagulation and propagating the reactions leading to fibrin formation. This endothelial cell-dependent pathway suggests a central role for factors VIII and IX consistent with their importance in hemostasis.The modern view of hemostasis and thrombosis was largely influenced by the publication 20 years ago of the waterfall (1) or cascade (2) theory of coagulation. This model has been enriched by addition of the platelet membrane, supplying a cellular surface promoting formation of the prothrombinase complex, which is a factor Va-Xa complex promoting efficient prothrombin activation (3-5). Endothelium, forming the luminal vascular surface, is another cell surface continuously interacting with coagulation factors. Traditionally, endothelium has been felt to play a passive role in hemostatic events providing an inert barrier to prevent exposure of coagulation factors and platelets to extravascular tissues. Recent studies have indicated that rather than providing an inert surface for the blood to flow over, the endothelial cell can play an active role in preventing activation of the coagulation system (6-9). Anticoagulant heparin-like molecules have been localized to the vessel surface (6). Antithrombin III bound to this heparin-like material on the endothelial cell surface demonstrates enhanced inactivation of pro...