Inflammatory mediators such as endotoxin, interleukin-l,6 (IL-lp) and tumor necrosis factor~ (TNF-=) dose-dependently incr~ the expression of tissue factor on the surface of cultured bovine aortic endothelial ¢¢11s (ABAE), human umbilical vein endothelial cells (HUVEC) and human monocytes. In ABAE, ¢ndotoxin-, IL-Ip-and TNFa-induced tissue factor expression was suppressed by interleukin-4 (IL-4) which also neutralized the pyrogeni¢ effect of ¢ndotoxin in HUVEC and moaocytes. IL-4 did not alter TN F-a-induced proeoagulant changes in HUVEC and monocytes but strongly protected the monoeyte surface against IL-I/~-induced proeoagulant changes. 1, INTRODUCTIONTissue factor (TF) is an ubiquitous membrane-anchored glycoprotein that initiates blood coagulation by forming a complex with circulating factors VI I and Vlla We investigated the effect of IL-4 on the expression of TF induced by various inflammatory mediators on the surface of endothelial cells and monocytes. MATERIALS AND METHODS CellsAdult bovine aortic endothelial cells (ABAE) (passage 7-10) were isolated and cultured as already d~cribed [7] Human ambilical vein endothelial cells (HUVEC) (passage 3-10) were isolated and cultured as described [8] in 96-well mlcroplates in F l ~-I lure'S medium supplemented with 10% fetal calf serum, endothelial cell growth factor (30/~g/ml) and heparin (100/tg/ml) (Sigma, France).Mononuclear cells were obtained from human heparinized blood as described by Boyum [9]. Cells were plated for 30 rain at 370C into 96-well microplates (10 s calls/well). Non-adherent ¢¢11s were then harvested and adherent monoeyte (5 x 10 ~ cells/well) were used for the assay. Determination of tLssue factor activity on the cellsProcoagulant activity was assayed according to $uprenant ¢tal. [10l. Briefly, adherent cells were incubated at 37"C in M-199 (without Phenol red) with ¢ndotoxin (LPS-lipopolysaccharide from E. coti strain: 055:B5) (Sigma, France), IL-I/~ or TNF~ (Teba, France) in the absence or presen~ of the indicated concentrations of IL-4 (Tebu, France). Incubation lasted for 18 h for ABAE and monoeytes and 6 h for HUVEC. The mediam was removed and wells were washed twice with 1 ml of phosphate-bulTered saline (PBS) and incubated for 45 rain at 37"C with 250 pl of M-199 containlng PPSB (0.44 U/ml FVII) (lntertran~fusion, France) and 100/t~/ml of substrate $2222 (Kabi, Sweden). The optical density (OD) was measured at 405 nm. The TF activity was obtained from a standard curve (log [~OD~o~/min] vs. log [U/roll) using serial dilations of rabbit brain thromboplastin in M-199 assayed as described above. Undiluted thromboplastin was arbitrarily assigned a value of I U/ml. The TF activity was normalized to the cell counts from the same well and expressed as,aU of TF/10 s ¢¢11s. RESULTS AND DISCUSSION Effect of LPS, IL-I~ and TNF-~ on TF expressionin ABAE, HUVEC and monocytes The addition of LPS, IL-I/~ or TNF-0t to adherent cells resulted in a time-dependent expression of TF on the cell surface (Fig. 1)

Endotoxin(LPS), mterleukm-1jI (IL-l) and tumor necrosis factor-a (TNF) mcreased the expression of tissue factor. a membrane-anchored glycoprotein that initiates blood coagulation on the surface of cultured bovine aortic endothelial cells (ABAE) and human monocytes. These compounds simultaneously reduced the amount of thrombomodulin on the endothelial cell surface. further contributing to the procoagulant activity of the endothelium or monocytes.On endothelial cells and monocytes, mterleukin-4 (IL-4) and mterleukm-13 (IL-13). a newly described lymphokine. both strongly inhibited LPS-induced tissue factor expression. a similar activity also being obtained with regard to the pyrogemc effects of IL-1 or TNF. When measured in parallel. IL-4 and IL-13 counteracted thrombomoduhn down-regulation induced by LPS. IL-1 or TNF m endothelial cells. These results therefore show that both IL-4 and IL-13 protect the endothehal and the monocyte surface agamst mflammatory mediator-induced procoagulant changes.

The platelet fibrinogen receptor GpIIb-IIIa is currently considered a target of choice for drugs used in the prevention and treatment of thrombosis. Ethyl 3-[N-[4-[4-[amino[(ethoxycarbonyl)-imino] methyl]phenyl]-1,3-thiazol-2-yl]-N-[1-[(ethoxycarbonyl)methyl] piperid-4-yl] amino]propionate (6, SR 121787) is a new antiaggregating agent which generates in vivo the corresponding diacid 19d (SR 121566), non-peptide GpIIb-IIIa antagonist. In vitro, 19d inhibited ADP-induced aggregation of human and baboon platelets (IC50 = 46 +/- 11 and 54 +/- 6 nM, respectively), and on human platelets, 19d antagonized the binding of 125I-labeled fibrinogen (IC50 = 19.2 +/- 6.2 nM). Ex vivo, 8 h after an i.v. administration of 19d (100 micrograms/kg, i.v.) to baboons, ADP-induced aggregation was strongly inhibited (more than 90%). At 8 h, the ED50 value was 24 +/- 3.3 micrograms/kg), and even 24 h after the administration of a single dose of 100 micrograms/kg of 19d, platelet aggregation was still significantly inhibited (50 +/- 6% inhibition, P < 0.05). In the same species, the oral administration of 500 micrograms/kg of 6 produced a nearly complete inhibition of aggregation for up to 8 h (ED50 at 8 h was 193 +/- 20 micrograms/kg). After an oral dose of 2 mg/kg of 6, an antiaggregating effect was still observed at 24 h (44 +/- 12% inhibition, P < 0.05). 6 was well tolerated in animals, showing that, on the basis of these studies, it is a suitable candidate for development as an orally active antithrombotic agent.

SSR182289A competitively inhibits human thrombin (K i ϭ 0.031 Ϯ 0.002 M) and shows good selectivity with respect to other human proteases, e.g., trypsin (K i ϭ 54 Ϯ 2 M), factor Xa (K i ϭ 167 Ϯ 9 M), and factor VIIa, factor IXa, plasmin, urokinase, tPA, kallikrein, and activated protein C (all K i values Ͼ250 M). In human plasma, SSR182289A demonstrated anticoagulant activity in vitro as measured by standard clotting parameters (EC 100 thrombin time 96 Ϯ 7 nM) and inhibited tissue factor-induced thrombin generation (IC 50 of 0.15 Ϯ 0.02 M). SSR182289A inhibited thrombin-induced aggregation of human platelets with an IC 50 value of 32 Ϯ 9 nM, but had no effect on aggregation induced by other platelet agonists. The anticoagulant effects of SSR182289A were studied by measuring changes in coagulation markers ex vivo after i.v. or oral administration in several species. In dogs, SSR182289A (0.1-1 mg/kg i.v. and 1-5 mg/kg p.o.) produced dose-related increases in clotting times. After oral dosing, maximum anticoagulant effects were observed 2 h after administration with increases in thrombin time, 2496 Ϯ 356%; ecarin clotting time (ECT), 1134 Ϯ 204%; and activated partial thromboplastin time (aPTT), 91 Ϯ 20% for the dose of 3 mg/kg p.o., and thrombin time, 3194 Ϯ 425%; ECT, 2017 Ϯ 341%; and aPTT, 113 Ϯ 9% after 5 mg/kg p.o. Eight hours after administration of 3 or 5 mg/kg SSR182289A, clotting times were still elevated. SSR182289A also showed oral anticoagulant activity in rat, rabbit, and macaque. Hence, SSR182289A is a potent, selective, and orally active thrombin inhibitor.

Sixty-five natural flavonoids of various chemical classes were screened for their ability to inhibit the procoagulant activity of adherent human monocytes stimulated by endotoxin and interleukin-1 beta in vitro. Eighteen of these compounds inhibited the interleukin-1 beta-induced expression of tissue factor on human monocytes, but the most active compound was a biflavonoid: hinokiflavone.