The garlic-derived organosulfur compound diallyl trisulphide suppresses tissue factor function.

Tissue factor (TF) is a critical initiator of extrinsic coagulation that sometimes causes thromboembolism. Diallyl trisulphide (DATS) is a secondary metabolite of allicin generated in crushed garlic, with various pharmacological effects. This study aimed to clarify the effect of DATS on the extrinsic coagulation elicited by TF and arteriosclerosis. TF activity was measured using a clotting assay in TF-expressing HL60 cells. DATS inhibited TF activity in a dose-dependent manner. TF expression in TNF-α-stimulated human umbilical vein endothelial cells was examined using real-time PCR and western blotting. DATS inhibited TF mRNA and protein expression induced by TNF-α via inhibition of JNK signalling. The effect of DATS on arteriosclerosis was also examined in apolipoprotein E-deficient mice. DATS administration in these mice tended to decrease atherosclerotic lesion size. These results strongly suggest that DATS prevents thromboembolism triggered by atherosclerosis via the inhibition of plaque formation and TF function.

Protective effect of Myrtle (Myrtus communis) on burn induced skin injury.

Thermal skin burns cause local injury as well as triggers acute systemic inflammation response where the imbalance between oxidative and antioxidative system occurs. As an alternative treatment, various medicinal herbs are used to treat burn injuries in many countries. In this study, the possible protective role of oral or topical Myrtle (Myrtus communis L.) treatment against burn-induced damage was investigated. The dorsum of the Wistar Albino rats was shaved and exposed to 90 °C water bath in burn group or 25 °C water bath in control group for 10 s under ether anesthesia. Myrtle extract was applied 100 mg/kg/day for 2 days either orally or topically. In skin samples; malondialdehyde and glutathione levels, catalase, superoxide dismutase, nitric oxide and tissue factor activities were determined. Skin tissues were also examined by light microscopy. Severe thermal skin burn injury caused a significant decrease in glutathione level, superoxide dismutase, catalase and tissue factor activities as well as nitric oxide level, which was accompanied with significant increases in skin malondialdehyde level. Myrtle treatment reversed all these biochemical indices except topical Myrtle treated group's nitric oxide level, as well as histopathological alterations, which were induced by thermal trauma. Both oral and topical Myrtle extract treatment was found to have protective role in the burn induced oxidative injury, which may be attributed to the potential antioxidant effect of Myrtle. As a conclusion, Myrtle significantly diminishes burn-induced damage in skin.

Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase blunts factor VIIa/tissue factor and prothrombinase activities via effects on membrane phosphatidylserine.

OBJECTIVE: 3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (statins) exhibit antithrombotic properties that are independent of reductions in circulating LDL cholesterol. We hypothesized that these antithrombotic properties are mediated by membrane alterations secondary to disrupted lipid metabolism. METHODS AND RESULTS: EA.hy926 cells were incubated in the presence of 1 micromol/L atorvastatin supplemented with fetal bovine serum or lipid-depleted serum mixtures. Lipid restriction alone had no effect on cell lipid composition but when atorvastatin was included, phosphatidylserine, sphingomyelin, and cholesterol were reduced by 50% while ceramide content decreased by 70%. These changes in lipid composition did not alter the association of decay accelerating factor or tissue factor with lipid rafts. Atorvastatin in combination with lipid restriction reduced factor VIIa/tissue factor activity by as much as 75% but did not alter tissue factor expression. Prothrombinase activity was reduced to an extent similar to factor VIIa/tissue factor. Mevalonic acid but not LDL reversed the observed changes in lipid content and prothrombinase activity induced by atorvastatin. These findings were confirmed in primary cells. CONCLUSIONS: Inhibition of HMG-CoA reductase limits exposure of phosphatidylserine at the cell surface by restricting the cellular pool of mevalonate-derived isoprenoids. This membrane alteration restricts the activity of proteolytic enzyme complexes that propagate the coagulation cascade.

The inhibitory effect of triterpenoid glycosides originating from Sanguisorba officinalis on tissue factor activity and the production of TNF-alpha.

We examined the inhibitory effects of novel triterpene glycoside compounds [ziyu-glycoside II (ZY-II) and its methyl ester (ZYM-201)], which originated from the roots of sanguisorba officinalis L. (Rosaceae), on tissue factor (TF) activity and tumor necrosis factor (TNF)-alpha production. In in vitro TF activity tests, ZY-II but not ZYM-201 strongly blocked lung TF activity with an IC50 value of 0.46 microM. By contrast, only ZYM-201 dose-dependently inhibited in vivo TF activity with an ED50 value of 1.7 mg/kg, when orally administered. Furthermore, ZYM-201 diminished both in vitro and in vivo TNF-alpha production with IC50 or ED50 values of 69.4 microM and 87.4 mg/kg, respectively. Therefore, these results suggest either that ZYM-201 may be developed as a potent inhibitor of both TF- and TNF-alpha-mediated diseases such as atherosclerosis and septic shock, or it may be a lead compound to be derivatized for further improvement of its curative efficacy.

Atorvastatin reduces thrombin generation after percutaneous coronary intervention independent of soluble tissue factor.

INTRODUCTION: Statins were previously shown to suppress cellular tissue factor (TF) in vitro. Here, we investigated the effect of atorvastatin on the TF-pathway and thrombin generation after coronary angioplasty and stenting in vivo. MATERIALS AND METHODS: A cohort of 30 patients with coronary artery disease (CAD) was randomised to treatment with either none (n=10), 10 mg (n=10) or 80 mg (n=10) atorvastatin per day for the postinterventional period of 6 months starting the day before percutaneous coronary intervention (PCI). Fasting blood samples were collected on admission and after 6 weeks and 6 months of statin therapy to determine sTF, free tissue factor pathway inhibitor (TFPI) and prothrombin fragment F1.2 by immunoassay. RESULTS: Soluble TF (sTF) significantly correlated with thrombin generation as measured by prothrombin fragment F1.2 at baseline. This correlation was lost 6 weeks and 6 months after initiation of statin therapy. In vivo, F1.2 was significantly lowered after 6 months of statin therapy by both, low dose (0 vs. 10 mg: 1.3+/-0.3 vs. 0.7+/-0.2 ng/ml; P<0.05) and high dose (0 vs. 80 mg: 1.2+/-0.3 vs. 0.6+/-0.2 ng/ml; P=0.01) atorvastatin compared to control. However, sTF and free TFPI did not change significantly with atorvastatin therapy when compared to baseline or control. CONCLUSIONS: Our results demonstrate reduced in vivo generation of thrombin six months after percutaneous coronary intervention and statin therapy independent of sTF and free TFPI.

Effect of kaikasaponin III obtained from Pueraria thunbergiana flowers on serum and hepatic lipid peroxides and tissue factor activity in the streptozotocin-induced diabetic rat.

We investigated the effect of kaikasaponin III (KS-III) on Phase I and II enzymes and tissue factor (TF) activity to elucidate the pharmacological actions of this immunosuppressive saponin in the diabetic rat. This compound was obtained from the flower of Pueraria thunbergiana (Leguminosae) by chromatographic isolation. This crude drug (Puerariae Flos) has been used as a therapeutic agent for diabetes mellitus in traditional Korean medicine. KS-III prolonged the bleeding time and plasma clotting time in streptozotocin (STZ)-treated rats and increased the TF activity, suggesting that this compound has anti-thrombosis activity in STZ-induced rats. It also inhibited the formation of malondialdehyde (MDA) and hydroxy radicals in serum and liver, but promoted superoxide dismutase (SOD) activity. Low MDA concentrations and low xanthine oxidase and aldehyde oxidase activities were observed in the KS-III-treated rats, suggesting that such Phase I enzyme activities are the major source of lipid peroxidation. However, KS-III increased Phase II enzyme activities such as SOD, glutathione peroxidase, and catalase, suggesting the activation of free radical-scavenging enzymes. These results suggest that KS-III may exhibit its hypoglycemic and hypolipidemic effects by up-regulating or down-regulating antioxidant mechanisms via the changes in Phase I and II enzyme activities.

Nicotinamide inhibits endotoxin-induced monocyte tissue factor expression.

BACKGROUND: Tissue factor (TF) is the main initiator of blood coagulation in vivo. Its increased expression on activated monocytes is associated with thrombotic complications and mortality in conditions such as sepsis, disseminated intravascular coagulation and coronary artery disease. OBJECTIVE: The effect of the vitamin B derivative nicotinamide on endotoxin-induced monocyte TF and CD11b expression, soluble interleukin(IL)-6, and clotting onset time (COT) was studied. METHODS: Experiments were conducted in human peripheral blood leukocyte suspensions and in whole blood from eight healthy volunteers. Free oscillating rheometry (measuring COT) and flow cytometry were applied to evaluate the effect of endotoxin on TF, CD11b, IL-6 and the overall coagulation response of plasma supplemented with activated autologous leukocytes. RESULTS: In response to endotoxin, there was an increase in IL-6, TF and CD11b expression and a procoagulant shift of COT. At 4 mmol L-1 nicotinamide, inhibition of TF expression and IL-6 and a normalization of COT were seen. At 16 mmol L-1 nicotinamide, CD11b decreased also. The level of monocyte TF expression correlated with the COT readings, and the endotoxin-induced procoagulant shift of COT could be totally inhibited by blocking TF with an inhibitory antibody. CONCLUSIONS: These results demonstrate the ability of nicotinamide to inhibit the activation of coagulation associated with endotoxemia. We have previously shown that nicotinamide exerts strong anti-inflammatory effects. Evidence is accumulating for nicotinamide to have a therapeutic potential in modulating disease states in which there is a profound activation of coagulation and inflammation, such as in sepsis and disseminated intravascular coagulation.

[Coagulation inhibitors in severe sepsis: state of the art]. / Inhibiteurs de la coagulation et états septiques graves.

OBJECTIVE: To present and discuss the rationale and the results of clinical trials using supplementation with physiologic anticoagulants (Tissue Factor Pathway Inhibitor (TFPI), AntiThrombin (AT), and Protein C (PC) in patients with severe sepsis. RATIONALE: An early activation of the coagulation cascade occurs in severe sepsis. TFPI, AT, and PC are major inhibitors of the coagulation cascade, and additionally modulate inflammatory and vascular reactions. They are consumed or inhibited in the sepsis pathologic process. Therapeutic supplementation with these inhibitors could improve the sepsis-induced organ failures and mortality. CLINICAL RESULTS: Randomized controlled studies were recently completed. No effect on the mortality rate could be documented after treatment with recombinant TFPI. AT concentrates neither improve mortality, but a biological interaction with heparin therapy could have biased the study results. Treatment with recombinant activated PC (alpha-drotrecogin) was associated with a significant reduction in the mortality rate of severely ill patients and received recently the approval from FDA and EC authorities in this indication. An increase in the rate of hemorrhagic adverse effects has been observed with these compounds, justifying a strict observance of contraindications and of patients selection. PROSPECTIVE: Additional studies are needed to give confirmation of the positive effects of activated PC supplementation in less severely ill patients, children and specific clinical situations. The effects of new anticoagulant compounds are currently evaluated in preclinical studies.

A new in vitro tissue factor inhibitory triterpene from the fruits of Chaenomeles sinensis.

Tissue factor (TF, tissue thromboplastin) accelerates the blood clotting, activating both the intrinsic and the extrinsic pathways to serve as a cofactor. In order to isolate TF inhibitors from the fruits of Chaenomeles sinensis, an activity-guided purification was carried out to yield four triterpenoid compounds. One compound was new and its structure was elucidated as 28-O-beta- D-glucopyranosyl-2alpha,3beta-dihydroxyolean-12-ene-24,28-dioic acid (2), named chaenomeloside A by means of spectral analysis and chemical conversion. Other compounds were trachelosperoside A-1 (1), oleanolic acid (3) and ursolic acid (4). Compound 2 and its aglycone 2a, named chaenomelogenin A inhibited by 50 % the TF activity at concentrations of 0.036 and 0.028 mM/unit of TF, respectively. Compound 1 isolated for the first time from this plant as well as 3 and 4 were inactive.

High doses of atorvastatin do not affect activity of prothrombinase in patients with acute coronary syndromes.

Membrane-dependent coagulation processes play a key role in acute coronary syndromes (ACS), where the generation of thrombin depends on the complex of activated factors X and V (prothrombinase complex) assembled on activated platelets. The aim of the present study was to evaluate prothrombinase activity in patients with ACS and to examine the effect of treatment with 80 mg/day atorvastatin on prothrombinase activity. Blood samples were obtained at admission from 22 patients with ACS, and then again at 2 weeks and at 16 weeks after double-blind randomization to either placebo or atorvastatin. Prothrombinase activity was evaluated by measuring the generation of thrombin by in vitro reconstructed thrombi, and also by measuring plasma levels of prothrombin fragment F1 + 2. Twenty age-matched subjects with stable angina and 11 without coronary disease were used as controls. At admission, prothrombinase activity and F1 + 2 were significantly higher in ACS patients than in controls. Prothrombinase activity was still high at 2 weeks while it returned to normal levels at 16 weeks. F1 + 2 remained high both at 2 and at 16 weeks. Our data indicate that prothrombinase activity is high in patients with ACS, and that it is not affected by high-dose atorvastatin.

Platelet aggregation through prothrombinase activation induced by non-aggregant doses of platelet agonists.

Activation of the prothrombinase complex, which catalyzes the formation of thrombin from prothrombin, is crucial for the (patho)physiological processes of hemostasis and thrombosis. We here report that washed platelets supplemented with prothrombin can be irreversibly aggregated with otherwise non-aggregant doses of adenosine diphosphate (10 micromol/l), thrombin (0.06 U/ml), or collagen (1 microg/ml). Prothrombinase-catalyzed prothrombin to thrombin conversion most probably supports this aggregation response, since inhibitors of thrombin (hirudin or heparin) and an inhibitor of activated factor X (DX-9065a) impair the response. A certain degree of agonist-induced platelet activation seems to be required for this prothrombin-supported aggregation response, since prothrombin alone does not induce aggregation, and blockade of glycoprotein Ia/IIa with a specific antibody inhibits the platelet aggregation response to collagen and prothrombin. These results may suggest that activation of the prothrombinase complex could be a common step of the platelet response to distinct agonists, which may be achieved at low levels of platelet stimulation.

No effect of methacrylate-based bone cement CMW 1 on the plasmatic phase of coagulation, red blood cells and endothelial cells in vitro.

The compatibility of a methacrylate-based bone cement (CMW 1, DePuy International Ltd, England) used for the fixation of joint prostheses was evaluated on plasma, an erythrocyte suspension and cultured human endothelial cells. The extract of the cement was tested, following 1 hour and 7 days of curing. After the contact in vitro of the extract with plasma, activated partial thromboplastin time, antithrombin III, thrombin-antithrombin complexes and fibrin degradation products were assayed. Hemolytic activity was tested by adding the cement extracts to a suspension of erythrocytes. After 4 hours of incubation at 37 degrees C, the hemoglobin concentration was determined on the supernatants by the colorimetric method. The effect of the cement on tissue factor and thrombomodulin production was evaluated on human umbilical vein endothelial cell cultures. Tissue factor was determined in cell lysates by enzyme immunoassay, following 4 hours' incubation of cultures with the cement extract. Thrombomodulin was assayed in cell lysates by enzyme immuno assay, after 24 hours' incubation with the cement extract. The response to all trans-retinoic acid (ATRA) was tested. The cement caused no significant modifications of the coagulation tests, had no hemolytic activity, did not determine tissue factor production and did not modify thrombomodulin, compared to the negative control. The response to stimulation with ATRA was similar to that of the negative control. We conclude that the cement extract does not affect the plasmatic phase of coagulation, has no effect on erythrocytes, does not induce the expression of procoagulant activity by endothelial cells and does not impair their antithrombotic property, within the limits of the tests performed.

Tissue factor activity in patients with systemic lupus erythematosus: association with disease activity.

OBJECTIVE: Tissue factor (TF) is the major intrinsic initiator of clotting. TF expression on monocytes has been associated with high titers of anticardiolipin antibodies (aCL) in patients with antiphospholipid syndrome (APS) with thrombosis. We investigated the influence of clinical factors on TF activity in blood from patients with systemic lupus erythematosus (SLE) and examined the relationship between aCL and TF. METHODS: In this cross sectional study, consecutive patients with SLE from one rheumatology clinic gave blood samples for measurement of TF activity, aCL, and Russell viper venom time. We assessed disease activity by measuring sedimentation rate, anti-dsDNA, and complement components C3 and C4, and measured clinical indices. Associations were investigated with the Wilcoxon rank-sum test and linear regression. RESULTS: Sixty-nine patients contributed blood samples. The median age was 38 years, and 10 of the SLE patients had a history of thrombosis. Patients with active arthritis had TF activity 1.3 times that in patients without arthritis (p = 0.028). Users of nonsteroidal antiinflammatory drugs (NSAID) had TF activity significantly lower than nonusers (p = 0.010). Patients with previous thrombosis had TF activity significantly lower than patients without thrombosis (p < 0.001). Overall, aCL and TF activity were not associated when we adjusted for these clinical factors. CONCLUSION: Arthritis, previous thrombosis, and use of NSAID significantly modified TF activity in patients with SLE. Unlike previous reports, we found no association between aCL titer and TF activity, which may be due to our adjusting for other important clinical factors.

The influence exerted by a restricted phospholipid microenvironment on the expression of tissue factor activity at the cell plasma membrane surface.

Phosphatidylserine (PhtdSer) is an anionic aminophospholipid necessary for the development of optimal tissue factor (TF) activity at the cell surface. This study investigates the implication of a restricted lipid environment with respect to PhtdSer availability on TF expression and activity. K562 cells, showing a reduced ability to externalize PhtdSer, were transfected with human TF cDNA. PhtdSer exposure and TF activity were examined in transfected cells and compared to monocytic THP-1 cells expressing constitutive and inducible TF or megakaryocytic HEL cells showing a high PhtdSer externalization potency. TF expression was evidenced by flow cytometry and its activity measured using functional assays. PhtdSer exposure was monitored by enzymatic prothrombinase assay. One clone (DC9) expressed a stable amount of TF antigen without global modification of its membrane status. Despite a noticeable TF expression level, clone DC9 presented only a weak TF activity even after ionophore stimulation. The apparent Km, relative to factor X (FX) activation by TF-factor VIIa (FVIIa) complex, was 335 nM versus 70 nM for THP-1 cells. The velocity of the reaction was found 3-fold slower in DC9 than THP-1 cells. Ionophore treatment resulting in slightly enhanced amounts of available PhtdSer abolished this difference. The DC9 clone appears suitable for further investigations on the biology of TF expressed at the surface of cells where the contribution of PhtdSer is significantly attenuated. Such cells should enable further assessment of the role of TF as a receptor coupled to intracellular signaling pathways and its fate during apoptotic cell death.

Nitric oxide inhibits tissue factor synthesis, expression and activity in human monocytes by prior formation of peroxynitrite.

OBJECTIVE: Nitric oxide (NO) has antithrombotic properties by regulating platelet function, whereas direct effects on plasmatic coagulation are rarely described. In sepsis and inflammation, when synthesis of NO, oxygen radicals and toxic metabolites is crucial, the expression of tissue factor (TF) on monocytes stimulated by lipopolysaccharides (LPS) induces intravascular coagulation. This study was performed to examine the influence of NO and the NO-dependent metabolite peroxynitrite on LPS-induced TF expression and activity in human monocytes. DESIGN: Experimental study. SETTING: Laboratory for cell biology. METHODS: Human peripheral blood mononuclear cells were isolated from buffy coats by gradient centrifugation. The NO-releasing compounds SIN1 and NOC18 were used under different conditions. TF antigen was assayed by flow cytometry, and its activity by a clotting assay. TF-mRNA was measured by reverse transcriptase polymerase chain reaction (RT-PCR-ELISA). MEASUREMENTS AND RESULTS: Whereas NOC18, a pure NO donor, had no effect, SIN1, releasing both NO and superoxide (O2-), reduced TF expression and activity in a dose- and time-dependent manner; superoxide dismutase (SOD) reversed the SIN1-mediated effect. Adding the O2(-)-deliberating system hypoxanthin/xanthin oxidase (which had no significant effect per se) to NOC18, or using the NO and O2- reaction product peroxynitrite resulted in a reduction of TF expression. RT-PCR-ELISA indicated upregulation of TF-mRNA by SIN1 with a peak at 500 microM; higher doses had less effect. CONCLUSION: These data demonstrate an influence of NO on LPS-induced TF expression in monocytes by prior formation of peroxynitrite; furthermore, the balance between NO and O2- seems to play a crucial role.

Haemorrhagic toxicity of a large dose of alpha-, beta-, gamma- and delta-tocopherols, ubiquinone, beta-carotene, retinol acetate and L-ascorbic acid in the rat.

Antioxidants occasionally have become prooxidants when a large amount was ingested. The haemorrhagic toxicity of butylated hydroxytoluene, a synthetic antioxidant, may involve such a mechanism. This study investigated whether haemorrhage is induced by overdoses of tocopherols, beta-carotene, ubiquinone or L-ascorbic acid, which are representative biological antioxidants. Male Jcl:SD rats (six rats/group) were fed d-alpha, d-beta, d-gamma or d-delta-tocopherols, ubiquinone Q-10, beta-carotene or retinol acetate at a level of 0.5%, or L-ascorbic acid at 5% in the diet for 7 days. Only two rats given retinol acetate died with lung haemorrhages. Haemorrhages were observed in five or six, six, one, one, one or one of six surviving rats given d-alpha, d-beta or d-gamma-tocopherols, ubiquinone Q-10, beta-carotene or retinol acetate, respectively (except for a retinol group in which four rats survived). Major haemorrhages were noted in the epididymis. In the alpha-, beta- and gamma-tocopherol, ubiquinone Q-10, beta-carotene or retinol acetate-treated groups, prothrombin and kaoline-activated partial thromboplastin time indices were 26-28, 37, 59, 42, 63 and 65% or 27-28, 35, 65, 38, 59 and 28%, respectively, of the control values. Only the prothrombin index was significantly decreased to 67% in delta-tocopherol-administered rates, whereas controls and those receiving L-ascorbic acid showed no signs of bleeding or coagulation defect. The same tendency was also seen in the decreasing effect on vitamin K-dependent blood coagulation factors. These results suggest that the four naturally occurring tocopherols have a tendency to cause haemorrhage in the order of alpha > beta > gamma > delta, and ubiquinone Q-10 and beta-carotene als0o have relatively strong and weak haemorrhagic effects, respectively, with regard to prothrombin and partial thromboplastin time indices.

Priming effect of adrenic acid (22:4(n-6)) on tissue factor activity expressed by thrombin-stimulated endothelial cells.

Tissue factor (TF) which initiates clotting process can be expressed by stimulated endothelial cells (EC). TF is an apolipoprotein requiring an association with phospholipids (PL) in order to become active. Also PL constitute an important storage pool of polyunsaturated fatty acids (PUFAs) in EC which can be modulated by diet or cell medium supplementation. In order to test the effect of such manipulation upon TF activity, we have pre-enriched human EC cultures with different fatty acids of nutritional interest. TF was evaluated after 4 h of thrombin stimulation by using a chromogenic method. Without additional stimulating agents, these acids have no effect on the basal level of TF. Eicosapentaenoic and docosapentaenoic acids appeared to be ineffective at the stimulated TF level. Only adrenic acid (22:4(n-6)) has been found to significantly enhance TF activity of thrombin-stimulated endothelial cells. Other TF inducers were also tested after 22:4(n-6) enrichment. An increase tendency of TF expression was found only with tumor necrosis factor, whereas interleukin-1 beta, lipopolysaccharide and especially phorbol myristate acetate stimulations were not significantly modified. The priming effect of adrenic acid on thrombin stimulated TF expression might involve alterations of signal transduction pathways rather than modifications of apolipoprotein III environment. Adrenic acid, which is a prostacyclin inhibitor, appears to be potential prothrombotic agent.