Angiotensin AT1 receptor antagonist irbesartan decreases lesion size, chemokine expression, and macrophage accumulation in apolipoprotein E-deficient mice.

Recent data suggest that angiotensin II AT1 receptor antagonists may be beneficial in the treatment of atherosclerosis. To clarify how AT1 receptor antagonists reduce atherosclerosis, the effect of irbesartan on atherosclerotic lesion development was determined in low-fat, chow-fed apolipoprotein (Apo) E-deficient mice. Irbesartan (50 mg/kg per day) strongly decreased lesion development after a 12-week treatment period (lesion size: irbesartan treated, 20,524 +/- 4,200 microm(2) vs. control, 99,600 +/- 14,500; 79.4% inhibition, p < 0.001). This effect was not due to an effect of irbesartan on lipoprotein levels because irbesartan slightly increased total cholesterol levels and decreased the ratio of Apo A-I relative to Apo B levels. Immunochemical analysis of the atherosclerotic lesions using the mac3 monoclonal antibody showed the presence of macrophages in the lesions of control mice, whereas sections from irbesartan-treated animals only showed occasional labeling in the lesion area. These data suggest that irbesartan inhibits monocyte/macrophage influx into the vessel wall. Therefore, expression levels of monocyte chemoattractant protein-1 (MCP-1), as well as other chemokines involved in macrophage infiltration into the lesion area, were measured in the aortic sinus of control and irbesartan-treated animals. Irbesartan treatment strongly decreased MCP-1 mRNA levels as well as MCP-1 immunostaining in the lesion area. This effect of irbesartan on MCP-1 occurred without an effect on CCR2, the receptor of MCP-1. Expression of macrophage inflammatory protein (MIP)-1alpha, another CC chemokine expressed in atherosclerotic lesions, was also reduced after irbesartan treatment, without effect on CCR3 and CCR5, the receptors of MIP-1alpha. Concomitantly, the expression of the angiogenic chemokines KC and MIP-2, which are functionally related to interleukin-8, were downregulated, whereas their shared receptor CXCR2 was upregulated. These data suggest that inhibition of the inflammatory component of lesion progression plays an important role in the inhibitory effect of AT1 receptor antagonists on atherosclerotic lesion formation.

The calcium inhibitor SR33805 reduces intimal formation following injury of the porcine carotid artery.

We studied the effect of SR33805, a calcium channel blocker, in vitro on the proliferation of vascular smooth muscle cells (SMC) stimulated by foetal calf serum, basic fibroblast growth factor and platelet derived growth factor, and in vivo with regard to SMC migration and proliferation which occurred following injury of the porcine carotid artery. The intimal lesion was induced by a silasten collar surgically positioned around the carotid artery and by a stenosis reducing blood flow by 50% for 30 days. Animals received SR33805 (5 mg/kg/day) 8 days before the induction of the lesion and up to 30 days after. In vitro, SR33805 inhibited in a dose-dependent manner growth factor-induced proliferation of SMC (0.20

Effect of clopidogrel on thrombin generation in platelet-rich plasma in the rat.

Clopidogrel (25 mg/kg, p.o.), a potent and selective inhibitor of ADP-induced platelet aggregation, significantly inhibited, in the presence of platelets, ex vivo thrombin generation triggered by low concentrations of tissue factor. Clopidogrel reduced the area under the curve (23%, p <0.05) and the thrombin peak concentration (35%, p <0.05) but did not affect the lag phase of thrombin generation. Under the same experimental conditions, heparin (100 microg/ml) inhibited thrombin generation mostly by delaying and by reducing the burst of thrombin. In a stasis-induced venous thrombosis model in rats under low thrombogenic challenge, clopidogrel inhibited thrombus formation (ED50 = 7.9+/-1.5 mg/kg, p.o. - n = 10), confirming the existence of a close relationship between platelet activation and thrombin generation leading to blood coagulation and venous thrombosis.

The antiaggregating and antithrombotic activity of clopidogrel is potentiated by aspirin in several experimental models in the rabbit.

It is unknown whether the addition of aspirin might increase both the efficacy and the potency of clopidogrel, a potent and selective ADP blocker. For that purpose, the efficacy of clopidogrel (1-20 mg/kg, p.o.) administered orally to rabbits alone or in combination with aspirin (0.1-10 mg/kg, p.o.) was determined in several experimental models. A potent synergistic effect of the clopidogrel/aspirin association was demonstrated with regard to collagen-induced platelet aggregation measured ex vivo. Similarly, aspirin potentiated the antithrombotic activity of clopidogrel measured with regard to experimental thrombosis induced by a silk thread or on stents placed in an arteriovenous shunt, thrombus formation following electrical stimulation of the rabbit carotid artery and with regard to 111In-labeled platelet deposition on a stent implanted in an arteriovenous shunt or on the subendothelium following air drying injury of the rabbit carotid artery. A similar potentiating effect of aspirin was obtained with regard to myointimal proliferation (restenosis) in the femoral arteries of atherosclerotic rabbits which occurred as a consequence of stent placement. The clopidogrel/aspirin combination showed only additive-type effects on bleeding time prolongation induced by ear transection in the rabbit, therefore showing that combined inhibition of cyclooxygenase and ADP's effects provide a marked enhanced antithrombotic efficacy. Such a combination may provide substantial protection against platelet aggregation leading to thrombotic occlusion at sites of endothelial injuries and coronary artery stenosis in humans.

Effector protease receptor 1 mediates the mitogenic activity of factor Xa for vascular smooth muscle cells in vitro and in vivo.

The binding of 125I-factor Xa to human aortic smooth muscle cell (SMC) monolayers was studied. At 4 degreesC, 125I-factor Xa bound to a single class of binding sites with a dissociation constant value of 3.6+/-0.7 nM and a binding site density of 11,720+/-1,240 sites/cell (n = 9). 125I-factor Xa binding was not affected by factor X, thrombin, or by DX9065, a direct inhibitor of factor Xa, but was inhibited by factor Xa (IC50 = 5.4+/-0.2 nM; n = 9) and by antibodies specific for the effector cell protease receptor 1 (EPR-1), a well-known receptor of factor Xa on various cell types. A factor X peptide duplicating the inter-EGF sequence Leu83-Leu88-(Gly) blocked the binding of 125I-factor Xa to these cells in a dose-dependent manner (IC50 = 110+/-21 nM). Factor Xa increased phosphoinositide turnover in SMCs and when added to SMCs in culture was a potent mitogen. These effects were inhibited by DX9065 and by antibodies directed against EPR-1 and PDGF. Increased expression of EPR-1 was identified immunohistochemically on SMCs growing in culture and in SMCs from the rabbit carotid artery after vascular injury. When applied locally to air-injured rabbit carotid arteries, antibodies directed against EPR-1 (100 mug/ artery) strongly reduced myointimal proliferation 14 d after vascular injury (65-71% inhibition, P < 0.01). DX9065 (10 mg/kg, subcutaneous) inhibited myointimal proliferation significantly (43% inhibition, P < 0.05). These findings indicate that SMCs express functional high affinity receptors for factor Xa related to EPR-1, which may be of importance in the regulation of homeostasis of the vascular wall and after vascular injury.

Coagulation factor Xa induces endothelium-dependent relaxations in rat aorta.

The relaxing effect of coagulation factor Xa on phenylephrine-contracted rat aortic rings was compared with the effect of thrombin and trypsin. All three proteases induced a dose-dependent relaxation in the presence of an intact endothelium. EC50 values were 3 +/- 1, 24 +/- 9, and 16 +/- 1 nmol/L for thrombin, trypsin, and factor Xa, respectively. Whereas thrombin induced rapid relaxations followed by partial recontraction, trypsin and factor Xa induced slower sustained effects. Factor Xa-induced relaxations were not affected by hirudin at high concentrations (1 mumol/L) but were abolished by DX9065A, a specific inhibitor of the catalytic activity of factor Xa. Furthermore, no relaxations to factor Xa could be elicited in the presence of the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (100 mumol/L), whereas relaxations were not altered in the presence of the inactive enantiomer N omega-nitro-D-arginine methyl ester (100 mumol/L). Addition of factor Xa together with thrombin induced relaxations that were larger than those induced by thrombin alone, whereas factor Xa had no additional effects on trypsin-induced relaxations. Further-more, factor Xa relaxed thrombin-desensitized aortic rings but was ineffective in trypsin-desensitized tissues. These data suggest that factor Xa acts on a cleavable endothelial receptor that induces NO release, resulting in the relaxation of precontracted rat aortic rings. Factor Xa does not act through endothelial thrombin receptors but may activate another cleavable trypsin-sensitive receptor.

Intimal hyperplasia following vascular injury is not inhibited by an antisense thrombin receptor oligodeoxynucleotide.

Thrombin is a multifunctional serine protease with central functions in hemostasis, but demonstration of its role in the initiation and maintenance of cell proliferation which occurs following vascular injury is still lacking. To determine the role played by thrombin and its receptor in neointimal accumulation of smooth muscle cells in a rabbit carotid artery model, we have used an 18 mer antisense phosphorothioate oligonucleotide (ODN) directed against the translation initiation region of the human thrombin receptor gene. The antisense ODN inhibited in a dose-dependent manner thrombin- or thrombin receptor activating peptide-induced human aortic smooth muscle cell proliferation. The growth-inhibitory effect of thrombin receptor antisense ODN was preventable by an excess of sense oligomer and specific for thrombin. The suppression of growth was accompanied by a marked decrease of the level of thrombin receptor expression as evidenced by [125I]-thrombin binding to smooth muscle cells. Under the same experimental conditions, the corresponding sense ODN was inactive. The effect of the antisense ODN on intimal smooth muscle hyperplasia in rabbit carotid arteries subjected to endothelial injury was then investigated. The topical application of the antisense (500 microg/artery) but not the sense ODN dissolved in F127 pluronic gel around the injured artery resulted, 2 weeks after the application, in a dramatic reduction of the expression of the thrombin receptor mRNA and protein levels as determined by in situ hybridization and immunohistochemistry. However, intimal smooth muscle cell accumulation as estimated by an intimal to medial cross-sectional area ratio was reduced only by 2.7% (vs. 10.3% for the sense ODN), whereas r-hirudin (200 microg/kg/day, s.c.), a potent direct thrombin inhibitor significantly reduced the formation of neointima in denuded carotid arteries (35.4% inhibition, P = 0.03).

Simvastatin inhibits myointimal hyperplasia following carotid artery injury in cholesterol-fed rabbits.

The effect of simvastatin, a potent inhibitor of 3-hydroxy 3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) was evaluated in an experimental model of myointimal hyperplasia in cholesterol-fed rabbits. Myointimal hyperplasia was induced by an air-drying injury of the left carotid artery followed by a 2%-cholesterol diet for 14 days. A 2-week oral treatment with simvastatin (6 mg/kg/day, p.o.) significantly lowered the circulating levels of cholesterol and triglycerides (41% and 49% inhibition respectively) as well as the low density lipoprotein (LDL)-cholesterol and high density lipoprotein (HDL)-cholesterol levels. Simvastatin also strongly affected the uptake of cholesterol in the arteries occurring as a consequence of vascular injury (44% inhibition, P < 0.001). Morphometric analysis revealed that both the intima and the media areas increased substantially 2 weeks after the lesion and showed a considerable smooth muscle cell accumulation in the neointima together with the presence of numerous foam cells. A 16-day oral treatment with simvastatin strongly reduced smooth muscle cells hyperplasia occurring in both the media and the intima following deendothelialization (19% and 60% inhibition respectively) suggesting that simvastatin may be a useful inhibitor of restenosis which occurs following vascular injury.

DX 9065A a novel, synthetic, selective and orally active inhibitor of factor Xa: in vitro and in vivo studies.

DX 9065A is the first member of a newly developed series of synthetic and selective inhibitors of factor Xa. DX 9065A inhibited in a dose-dependent manner human factor Xa with K iota value of 3.1 +/- 0.5 nM. Steady-state studies revealed that DX 9065A was a competitive inhibitor of factor Xa. DX 9065A inhibited thrombin generation occurring via both the extrinsic and intrinsic pathway in vitro and in vivo. After i.v. injection to rabbits, DX 9065A displayed prolonged anti-factor Xa activity and inhibition of thrombin generation. Pretreatment of mice with DX 9065A dose-dependently improved the survival rate of mice injected with a lethal dose of tissue factor (ED50 = 1.1 +/- 0.2 mg/kg). After p.o. administration, DX 9065A caused a reduction in tissue factor-induced mortality of mice with ED50 value of 56 +/- 7 mg/kg. When given i.v. to rats, DX 9065A exhibited a dose-dependent antithrombotic effect against factor Xa + stasis-induced venous thrombosis (ED50 = 1.2 +/- 0.7 mg/kg i.v.), but also in an arteriovenous shunt thrombosis model (ED50 = 8.1 +/- 3.5 mg/kg i.v.) without affecting bleeding time significantly. Similar effects were obtained after s.c. or p.o. administration. In rabbits, after i.v., s.c. or p.o. administration, DX 9065A inhibited stasis-induced thrombosis after injection of tissue factor with ED50 values of 0.03 +/- 0.01, 0.3 +/- 0.07 and 50.5 +/- 19 mg/kg, respectively (n = 10). DX 9065A inhibited in a dose-dependent manner endotoxin-induced venous thrombosis in the rabbit (ED50 = 0.25 +/- 0.1 mg/kg i.v.) (n = 5) and reduced the decrease in platelet number and circulating fibrinogen levels in an experimental model of tissue factor-induced disseminated intravascular coagulation. Compared to standard heparin, DX 9065A exhibited a favorable antithrombotic/bleeding ratio, therefore showing that it might be considered as a promising compound in the treatment and prevention of various thrombotic diseases.

Platelet-activating factor (PAF) is not an essential component of the cascade leading to smooth muscle cell proliferation following vascular injury.

In order to evaluate the relative importance platelet-activating factor (PAF) in the proliferative process leading to restenosis, the effect of SR 27417, a novel highly potent PAF receptor antagonist, on PAF-induced rabbit aortic smooth muscle cell (SMC) proliferation and intimal hyperplasia in rabbit carotid arteries subjected to air-drying endothelial injury was investigated. When added to low concentrations of foetal calf serum, PAF showed a dose-dependent mitogenic effect with regard to rabbit arterial SMC. SR 27417 inhibited PAF-induced SMC growth (IC50 = 2.4 +/- 0.4 nM) but remained without effect on the mitogenic effect of foetal calf serum. A 16 day treatment of SR 27417 (10 mg/kg per day, p.o.) abrogated PAF-induced platelet aggregation ex vivo but did not affect the development of intimal thickening, therefore showing that PAF is not an essential component of the cascade leading to restenosis following vascular injury.

Effect of SR 33805 on arterial smooth muscle cell proliferation and neointima formation following vascular injury.

The possible activity of SR 33805 ([[N-[dimethoxy-3,4-phenethyl]-N- methylamino-propoxyl]-4-benzenesulfonyl]-2-isopropyl-3-methyl-1-in dole), a novel Ca2+ channel blocker, in early atherogenesis was investigated. In vitro, SR 33805 strongly inhibited fetal calf serum-induced proliferation of cultured human aortic smooth muscle cells with an IC50 value of 0.3 +/- 0.1 microM (n = 3). In this respect, SR 33805 was several fold more active than the reference compounds: diltiazem, verapamil, nifedipine and fantofarone. SR 33805 was also a potent inhibitor of platelet-derived growth factor- or basic fibroblast growth factor-induced proliferation of human smooth muscle cells. SR 33805 inhibited serum-stimulated 45Ca2+ uptake in these cells, with an IC50 value of 47 +/- 18 nM. The effect of SR 33805 on intimal smooth muscle hyperplasia in rabbit carotid arteries subjected to air-drying endothelial injury was then investigated. After a 16-day treatment, SR 33805 (6.0 mg/kg/day p.o.) inhibited the development of intimal thickening. Under the same experimental conditions, nifedipine, verapamil, diltiazem (2 x 6 mg/kg/day p.o.--16 days) and fantofarone (12 mg/kg/day p.o.--16 days) were inactive. These results show that SR 33805, a novel and potent Ca2+ channel blocker, can reduce myointimal thickening following endothelial injury.

Cross-reactivity of human molecular markers for detection of prethrombotic states in various animal species.

The aim of the present study was to investigate the reactivity of immunoreagents developed for clinical applications in humans in different animal species (hen, mouse, rat, rabbit, guinea-pig, dog, pig, sheep, baboon). Prothrombin fragment 1 + 2, thrombin-antithrombin III complex and fibrinopeptide A were tested for coagulation, platelet factor 4 and beta-thromboglobulin for platelet activation, glycoprotein IIb-IIIa, glycoprotein Ib and P-selectin for platelet membrane glycoproteins, D-dimers for fibrinolysis, thrombomodulin for activation of endothelial cells and thrombospondin and von Willebrand factor for adhesive proteins. Prothrombin fragment 1 + 2, platelet factor 4, beta-thromboglobulin and D-dimers were revealed only in baboons. Fibrinopeptide A was well detected in baboons but weakly in mice, dogs, pigs and sheep. Whereas glycoprotein IIb-IIIa was revealed on guinea-pig, dog and sheep platelets and glycoprotein Ib on rabbit and dog platelets, P-selectin and thrombomodulin were never detected. Thrombospondin was revealed in hens, mice, rats, guinea-pigs, pigs, sheep and baboons and von Willebrand factor in mice, rats, guinea-pigs, dogs, pigs, sheep and baboons. Interestingly, thrombin-antithrombin III complex (TAT) was detected in all species tested except the hen. A time- and dose-dependent increase in TAT was observed when rats, dogs or pigs were infused with thromboplastin (4.5-450 microliters/kg/h), while administration of hirudin (1 mg/kg) abolished this TAT generation. Thus, the TAT immunoassay could provide a tool for the screening of antithrombotic drugs in a number of animal species. However, the possibility of using a wider panel of human immunoreagents would appear to be restricted to baboons which display good species cross-reactivity.

Comparison of a low-molecular-weight heparin (nadroparin calcium) and unfractionated heparin as adjunct to coronary thrombolysis with alteplase and aspirin in dogs.

BACKGROUND: Low-molecular-weight heparins may have a higher benefit to risk ratio than unfractionated heparin in preventing perioperative thrombosis. The antithrombotic effects of low-molecular-weight heparins, given as adjunctive therapy to alteplase and aspirin, have not previously been compared with those of unfractionated heparin in experimental models of coronary artery thrombosis. METHODS: Occlusive coronary thrombosis was induced in 5 groups of 10 dogs by placing a copper coil into the left anterior descending coronary artery. After 1 h of occlusion, intravenous alteplase (0.1 mg/kg bolus followed by 0.01 mg/kg/min for 30 min), and aspirin (bolus of 5 mg/kg) were administered in combination with one of the following study treatments given intravenously for 2 h: placebo (group 1); unfractionated heparin (200 IU/kg bolus followed by 100 IU/kg/h, group II); the low-molecular weight heparin, nadroparin calcium, in three different doses (100 IU/kg bolus followed by 50 IU/kg/h, group III; 200 IU/kg bolus followed by 100 IU/kg/h, group IV; and 300 IU/kg followed by 150 IU/kg/h, group V). Coronary patency was assessed with angiography at 10 min intervals and hemostasis parameters were measured at baseline, after 1 h of occlusion, and 30 and 120 min after commencing drug administration. RESULTS: Optimal reperfusion [Thrombolysis in Myocardial Infarction (TIMI) flow grade 3 without reocclusion] was more frequently observed in groups II (6/10), IV (8/10) and V (9/10) than in groups I (1/10) and III (3/10) (P < 0.05). Groups II and IV had similar patency rates (P = NS) and were therefore assumed to represent equivalent antithrombotic doses. Both nadroparin calcium and unfractionated heparin effectively prevented new thrombin generation as shown by repeated measurements of thrombin-antithrombin III complex levels in plasma. At equivalent antithrombotic doses, nadroparin calcium (group IV) was associated with significantly lower steady state values than standard heparin (group II) for activated partial thromboplastin time (41.3 +/- 48.9 versus 134.7 +/- 61.6 s), anti-Xa levels (2.4 +/- 0.5 vs 3.4 +/- 0.9 U/ml) and anti-IIa levels (0.8 +/- 0.1 versus 2.1 +/- 0.7 U/ml). CONCLUSION: Both nadroparin calcium and unfractionated heparin significantly enhance alteplase-induced thrombolysis in aspirin-treated dogs. At equivalent antithrombotic doses, nadroparin calcium was associated with less prolongation of the activated partial thromboplastin time and lower steady-state anti-Xa and anti-IIa activities.

Effect of SR-49059, a vasopressin V1a antagonist, on human vascular smooth muscle cells.

The effects of SR-49059, a new nonpeptide and selective arginine vasopressin (AVP) V1a antagonist, were investigated in binding and functional studies on cultured human aortic vascular smooth muscle cells (VSMC). Characterization of human vascular V1a receptors, using a specific V1a radioiodinated ligand, showed that [125I]-linear AVP antagonist binding to human VSMC membranes was time dependent, reversible, and saturable. A single population of high-affinity binding sites (apparent equilibrium dissociation constant = 15 +/- 6 pM; maximum binding density = 36 +/- 5 fmol/mg protein, i.e., approximately 3,000 sites/cell) with the expected V1a profile was identified. Exposure of these cells to AVP dose-dependently produced cytosolic free [Ca2+] increase [AVP concentration required to obtain a half-maximal response (EC50) = 23 +/- 9 nM] and proliferation (EC50 = 3.2 +/- 0.5 nM). SR-49059 strongly and stereospecifically inhibited [125I]-linear AVP antagonist binding to VSMC V1a receptors [inhibition constant (Ki) = 1.4 +/- 0.3 nM], AVP-evoked Ca2+ increase [concentration of inhibitor required to obtain 50% inhibition of specific binding (IC50) = 0.41 +/- 0.06 nM], and the mitogenic effects induced by 100 nM AVP (IC50 = 0.83 +/- 0.04 nM). OPC-21268, another nonpeptide V1a antagonist, was more than two orders of magnitude less potent than SR-49059 in these models. However, the consistent affinity (Ki = 138 +/- 21 nM) and activity found with OPC-21268 on human VSMC in comparison with the inactivity already observed for other human V1a receptors (liver, platelets, adrenals, and uterus) strongly suggested the existence of human AVP V1a-receptor subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of SR 47436, a novel angiotensin II AT1 receptor antagonist, on human vascular smooth muscle cells in vitro.

Proliferation of smooth muscle cells within the intima plays a key role in vascular occlusive disorders such as atherosclerosis and restenosis following balloon angioplasty. Among the factors that may be important in the development of vascular lesions, several authors have reported that the local angiotensin system participates in modulating the proliferation of smooth muscle cells after arterial injury. This study was therefore designed to characterize the antagonistic properties and to investigate the antiproliferative effect of a newly developed non-peptide angiotensin II AT1 receptor antagonist, SR 47436. This compound is a potent and competitive antagonist of the binding of [125I]angiotensin II to its receptor on cultured human aortic smooth muscle cells, exhibiting an IC50 value of 1.7 +/- 0.6 nM. SR 47436 was 10-fold more potent than DuP 753 (Losartan) (IC50 = 20.8 +/- 3.7 nM). In these same cells, SR 47436 potently inhibited the angiotensin II-induced [Ca2+]i increase (IC50 = 0.53 +/- 0.13 vs. 7.4 +/- 1.3 nM for DuP 753). Angiotensin II is a potent mitogen for human aortic smooth muscle cells in culture, exhibiting a maximum proliferative response at 1 microM. SR 47436 and Losartan prevented angiotensin II-induced proliferation of these cells in a dose-dependent manner (IC50 = 0.32 +/- 0.09 and 0.71 +/- 0.08 microM, respectively). SR 47436 displayed a marked in vitro inhibition of serum-induced smooth muscle cell proliferation (IC50 = 5.5 +/- 0.8 microM). A selective AT2 receptor antagonist, PD 123177 did not affect angiotensin II-induced responses in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue-type plasminogen activator is a potent mitogen for human aortic smooth muscle cells.

Tissue-type plasminogen activator (t-PA) is a potent and efficacious mitogen for growth-arrested cultured human aortic smooth muscle cells, stimulating an increase in cell number at 0.3-30 nM concentration. Double-chain t-PA is as efficient as single-chain t-PA in stimulating smooth muscle cell mitogenesis, whereas single-chain urokinase-type plasminogen activator (u-PA) or u-PA and plasmin or plasminogen are ineffective. Plasminogen activator inhibitor-1, Pefabloc-TPA, diisopropyl fluorophosphate or alpha 1-anti-trypsin inhibit the mitogenic effect of t-PA for smooth muscle cells in a dose-dependent manner, showing that it is dependent on the enzymatic activity. t-PA activated phosphoinositide turnover in smooth muscle cells through a pertussis toxin-insensitive pathway and stimulated proto-oncogene c-fos and c-jun mRNA levels. These findings indicate that t-PA stimulates vascular human smooth muscle cell proliferation and suggest for the first time that it may contribute to intimal smooth muscle cell proliferation after vascular injury as a result of angioplasty or vascular compromise during atherogenesis.

Chelerythrine, a selective protein kinase C inhibitor, counteracts pyrogen-induced expression of tissue factor without effect on thrombomodulin down-regulation in endothelial cells.

Endotoxin, interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) dose-dependently increased the expression of tissue factor and at the same time induced thrombomodulin down-regulation on the surface of cultured bovine aortic endothelial cells. Chelerythrine, a selective protein kinase C inhibitor, strongly reduced endotoxin-, IL1 beta- and TNF alpha-induced tissue factor expression but remained without effect with regard to thrombomodulin down-regulation measured in parallel. On the contrary, staurosporine, a highly potent, non-selective PKC inhibitor, simultaneously abolished tissue factor expression and thrombomodulin down-regulation induced by endotoxin, IL1 beta and TNF alpha. These results show that protein kinase C is deeply involved in the process leading to pyrogen-induced tissue factor expression and suggest that thrombomodulin down-regulation is regulated by a different pathway.

IL-4 and IL-13 exhibit comparable abilities to reduce pyrogen-induced expression of procoagulant activity in endothelial cells and monocytes.

Endotoxin (LPS), interleukin-1 beta (IL-1) and tumor necrosis factor-alpha (TNF) increased 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, interleukin-4 (IL-4) and interleukin-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 pyrogenic effects of IL-1 or TNF. When measured in parallel, IL-4 and IL-13 counteracted thrombomodulin down-regulation induced by LPS, IL-1 or TNF in endothelial cells. These results therefore show that both IL-4 and IL-13 protect the endothelial and the monocyte surface against inflammatory mediator-induced procoagulant changes.

ADP-dependence of platelet activation induced by a thrombin receptor agonist.

The synthetic peptide SFLLRNPNDKYEPF (Thrombin Receptor Agonist: TRA) has recently been shown to mimic the new amino-terminus created by cleavage of the thrombin receptor on platelets and therefore to act as a receptor agonist. In the present work, this peptide proved a potent aggregating agent for human platelets with an ED50 of 3 microM. The ADP removing enzyme system creatine phosphate/creatine phosphokinase (CP/CPK) and the ADP receptor antagonist ATP alpha S strongly inhibited platelet aggregation in response to low doses of TRA, indicating that TRA-induced platelet aggregation, like thrombin-induced aggregation is an ADP mediated event. CP/CPK had however no effect on aggregation in response to the agonist peptide at higher concentrations. Similar results were obtained with regard to the influence of TRA and thrombin on platelet adenylyl cyclase activity, while both agents induced nucleotide release from platelets in a dose-dependent manner. These results confirm that the aggregating effect of alpha-thrombin on human platelets is closely linked to its esterolytic activity at the receptor level and show that this aggregation is an ADP mediated event.

Antithrombotic activity of SR 46349, a novel, potent and selective 5-HT2 receptor antagonist.

SR 46349 (trans-4-[(3Z)3-(2-dimethylaminoethyl)oxyimino-3(2-fluorophe nyl) propen-1-yl] phenol, hemifumarate) is the first member of a newly-developed 5-HT2 antagonist series. SR 46349 potently inhibited serotonin-induced aggregation of rabbit and human platelets (IC50 = 1 and 3.9 nM respectively) but had no effect on the action of other platelet aggregating agents. SR 46349 was 118 and 25 times more potent than ketanserin against 5-HT+epinephrine-induced aggregation of rabbit and human platelets respectively. A single per os administration of SR 46349 (1 mg/kg) resulted in a strong inhibition of 5-HT+epinephrine-induced platelet aggregation in the rabbit as measured ex vivo (67% inhibition, 6 h after the administration). Intravenous or oral administration of SR 46346 inhibited in a dose-dependent manner venous thrombosis induced by ligature of the jugular vein of rabbits whose blood was made hypercoagulable by i.v. administration of tissue thromboplastin. The doses of SR 46349 which inhibited 50% of thrombus formation were 1.5 +/- 0.8 mg/kg and 17 +/- 0.5 mg/kg after i.v. or oral administration respectively. When given i.v. to rabbits, SR 46349 exhibited a dose-dependent antithrombotic effect in an arterio-venous shunt model. Significant increase of the bleeding time was observed after the i.v. administration of 5 mg/kg of SR 46349 (3-fold increase). In dogs, SR 46349 inhibited cyclic coronary artery blood flow variations, complete abolition of CFVs being achieved after the i.v. administration of 0.5 mg/kg. In conclusion, SR 46349 is a highly potent, selective antagonist of serotonin in vitro and is to be considered as a potent, orally active antithrombotic agent.