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.

Aryl Hydrocarbon Receptor Activation and Tissue Factor Induction by Fluid Shear Stress and Indoxyl Sulfate in Endothelial Cells.

Endogenous agonists of the transcription factor aryl hydrocarbon receptor (AHR) such as the indolic uremic toxin, indoxyl sulfate (IS), accumulate in patients with chronic kidney disease. AHR activation by indolic toxins has prothrombotic effects on the endothelium, especially via tissue factor (TF) induction. In contrast, physiological AHR activation by laminar shear stress (SS) is atheroprotective. We studied the activation of AHR and the regulation of TF by IS in cultured human umbilical vein endothelial cells subjected to laminar fluid SS (5 dynes/cm2). SS and IS markedly increased the expression of AHR target genes PTGS2 (encoding for COX2), AHRR, CYP1A1, and CYP1B1, as well as F3 (encoding for TF), in an AHR-dependent way. IS amplified SS-induced TF mRNA and protein expression and upregulation of AHR target genes. Interestingly, tyrosine kinase inhibition by genistein decreased SS- but not IS-induced TF expression. Finally, the increase in TF expression induced by laminar SS was not associated with increased TF activity. In contrast, IS increased TF activity, even under antithrombotic SS conditions. In conclusion, IS and SS induce AHR activation and AHR-dependent TF upregulation by different mechanisms. Impairment of the antithrombotic properties of shear stressed endothelium by toxic AHR agonists could favor cardiovascular diseases in CKD.

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.

Relevance of hemolysis-induced tissue factor expression on monocytes in soft clot formation in alcohol-containing blood.

The fluidity of cadaveric blood is an important characteristic in the post-mortem examination of cases of asphyxial death. Although it is empirically known that soft blood clots are present in cadaveric blood containing alcohol, the relationship between such clots and blood alcohol is unclear. We addressed this issue through in vitro studies using blood collected from healthy volunteers. Assessment of global hemostasis by rotational thromboelastometry revealed that ethanol treatment enhanced the procoagulant activity of whole blood. However, ethanol inhibited epinephrine-induced platelet aggregation, whereas plasma levels of von Willebrand factor and the activity of coagulation factors VIII and IX were unaffected. In contrast, tissue factor (TF) activity was higher in plasma obtained from ethanol-treated whole blood than that in plasma from untreated blood. Ethanol induced hemolysis of red blood cells, and the consequent hemoglobin (Hb) release promoted de novo synthesis of TF in isolated monocytes, as determined by real-time reverse transcription PCR, western blotting, and flow cytometry. However, ethanol itself did not induce TF expression in monocytes. Given that TF activates the extrinsic coagulation pathway and amplifies hemostatic reactions, Hb-induced TF expression in monocytes might contribute to soft blood clot formation.

[Effect of Agkistrodon Acutus Venom PCA on HUVEC Activity].

OBJECTIVE: To investigate the effects of Agkistrodon acutus venom protein C activator(PCA) on ultrastructure of human umbilical vein endothelial cells(HUVEC), and the levels of tissue factor(TF), vascular von Willebrand factor (vWF) and endothelin-1 secreted by HUVEC and to clarify the anti-thrombotic mechanism of PCA. METHODS: The experiments were divided into control group(DMEM), LPS group (LPS 0.1 µg/ml), PCA group(PCA 1 µg/ml) and PCA+LPS group (1 µg/ml PCA+ 0.1 µg/ml LPS). The morphology of endoplasmic reticulum, mitochondria and the number of autophagosome in HUVEC were observed by transmission electron microscopy. The TF, vWF and ET-1 were measured in the medium of each group by ELISA; RT-PCR was used to detect mRNA expression level of vWF and ET-1 in cells; and the protein expression level of TF in cells was detected by Western blot. RESULTS: Compared with the control group, the ultrastructural changes of HUVEC in the LPS group included the cell membrane getting rough, swelling of mitochondria and endoplasmic reticulum, and autophagosome increase, however, the ultrastructure differences between PCA and control group were not significant. Compared with the ultrastructure of HUVECs in LPS group, the swelling of mitochondria and endoplasmic reticulum disappeared in the LPS+PCA group, and the number of autophagosome decreased obviously. Compared with the control group, the content of ET-1, vWF and TF in cell culture supernatant, and the protein expression level of vWF, ET-1 gene and TF protein were significantly increased in LPS group (P<0.05); the expression levels of the 3 factors in the cell culture supernatant and cells in PCA group were not significantly different from the control group (P>0.05). The expression levels of TF, vWF and ET-1 in LPS group were significantly lower than those in LPS+PCA group (P<0.05). CONCLUSION: PCA(1 µg/ml) can reduce the ultrastructural changes of HUVEC induced by LPS, and inhibit the increase of TF, vWF and ET-1 secretion from HUVEC induced by LPS.

α-1 Antitrypsin Enhances Islet Engraftment by Suppression of Instant Blood-Mediated Inflammatory Reaction.

Islet cell transplantation has limited effectiveness because of an instant blood-mediated inflammatory reaction (IBMIR) that occurs immediately after cell infusion and leads to dramatic ß-cell death. In intraportal islet transplantation models using mouse and human islets, we demonstrated that α-1 antitrypsin (AAT; Prolastin-C), a serine protease inhibitor used for the treatment of AAT deficiency, inhibits IBMIR and cytokine-induced inflammation in islets. In mice, more diabetic recipients reached normoglycemia after intraportal islet transplantation when they were treated with AAT compared with mice treated with saline. AAT suppressed blood-mediated coagulation pathways by diminishing tissue factor production, reducing plasma thrombin-antithrombin complex levels and fibrinogen deposition on islet grafts, which correlated with less graft damage and apoptosis. AAT-treated mice showed reduced serum tumor necrosis factor-α levels, decreased lymphocytic infiltration, and decreased nuclear factor (NF)-κB activation compared with controls. The potent anti-inflammatory effect of AAT is possibly mediated by suppression of c-Jun N-terminal kinase (JNK) phosphorylation. Blocking JNK activation failed to further reduce cytokine-induced apoptosis in ß-cells. Taken together, AAT significantly improves islet graft survival after intraportal islet transplantation by mitigation of coagulation in IBMIR and suppression of cytokine-induced JNK and NF-κB activation. AAT-based therapy has the potential to improve graft survival in human islet transplantation and other cellular therapies on the horizon.

The Antithrombotic Potential of Tinzaparin and Enoxaparin Upon Thrombin Generation Triggered In Vitro by Human Ovarian Cancer Cells IGROV1.

BACKGROUND: A documented relationship between ovarian cancer and thrombosis does exist. Low-molecular-weight heparins (LMWHs) are cornerstone drugs in the primary prevention and treatment of venous thromboembolic events in patients with cancer. However, cancer cells may alter the efficiency of these antithrombotic agents. OBJECTIVE: We aimed to characterize the procoagulant phenotype of human epithelial ovarian adenocarcinoma cells, IGROV1, and to compare the capacity of tinzaparin and enoxaparin to inhibit thrombin generation triggered by these cells. METHODS: Thrombin generation induced by different concentrations of IGROV1 cells on platelet poor plasma (PPP) was assessed by the calibrated automated thrombogram assay. Tissue factor (TF) expression was studied using Western blot analysis. Then, the experimental model of thrombin generation was used to compare the inhibitory effect of clinically relevant concentrations of both tinzaparin and enoxaparin. The inhibitory concentration 50 (IC50) of the mean rate index and the endogenous thrombin potential and the 2-fold increase in lag time were analyzed on the basis of the anti-Xa and anti-IIa activities of the LMWHs. RESULTS: IGROV1 cells suspended into PPP resulted in a significant increase in thrombin generation in the absence of any exogenous source of TF and phospholipids. Tissue factor was expressed by IGROV1 cells. Tinzaparin was a more potent inhibitor of thrombin generation than enoxaparin. The inhibition of thrombin generation induced by IGROV1 cancer cells depended mainly on the anti-Xa activity of the LMWHs. CONCLUSION: This experimental study in ovarian cancer cells demonstrates that the antithrombotic activity of LMWHs is not completely predicted by the anti-Xa or anti-IIa activities measured in PPP.

Tissue factor expression in obese type 2 diabetic subjects and its regulation by antidiabetic agents.

OBJECTIVE: Increased coagulation activation may contribute to the high incidence of cardiovascular complications observed in obese and type 2 diabetes (T2D) subjects. Although tissue factor (TF), the primary initiator of coagulation is increased in obesity, its expression in adipose tissues and its association with metabolic parameters are unclear. We sought to compare TF expression in plasma and adipose tissues of obese subjects with and without T2D, its correlation with metabolic parameters, and regulation in response to antidiabetic drugs. METHODS: Subjects were recruited from diabetes clinics and adipose tissue was obtained by needle biopsy of lower subcutaneous abdominal depot. For the intervention study, subjects were randomized into treatment groups with rosiglitazone or metformin for 4 months. RESULTS: Plasma TF antigen, activity, and adipose TF mRNA were greater in obese T2D subjects compared with obese nondiabetics. Plasma TF activity correlated with fasting insulin, glucose, and free fatty acids, (FFAs), and adipose TF mRNA correlated with plasma FFA. Plasma TF activity was reduced by metformin and increased with rosiglitazone treatment. CONCLUSIONS: Specific diabetes-related metabolic parameters, but not obesity per se, are correlated with TF expression. Regulation of TF activity by different classes of antidiabetic drugs may relate to protective or adverse cardiovascular outcomes.

Determination of FVIIa-sTF Inhibitors in Toxic Microcystis Cyanobacteria by LC-MS Technique.

The blood coagulation cascade involves the human coagulation factors thrombin and an activated factor VII (fVIIa). Thrombin and fVIIa are vitamin-K-dependent clotting factors associated with bleeding, bleeding complications and disorders. Thrombin and fVIIa cause excessive bleeding when treated with vitamin-K antagonists. In this research, we explored different strains of toxic Microcystis aeruginosa and cyanobacteria blooms for the probable fVIIa-soluble Tissue Factor (fVIIa-sTF) inhibitors. The algal cells were subjected to acidification, and reverse phase (ODS) chromatography-solid phase extraction eluted by water to 100% MeOH with 20%-MeOH increments except for M. aeruginosa NIES-89, from the National Institute for Environmental Studies (NIES), which was eluted with 5%-MeOH increments as an isolation procedure to separate aeruginosins 89A and B from co-eluting microcystins. The 40%-80% MeOH fractions of the cyanobacterial extract are active against fVIIa-sTF. The fVIIa-sTF active fractions from cultured cyanobacteria and cyanobacteria blooms were subjected to liquid chromatography-mass spectrometry (LC-MS). The 60% MeOH fraction of M. aeruginosa K139 exhibited an m/z 603 [M + H]⁺ attributed to aeruginosin K139, and the 40% MeOH fraction of M. aeruginosa NIES-89 displayed ions with m/z 617 [M - SO3 + H]⁺ and m/z [M + H]⁺ 717, which attributed to aeruginosin 89. Aeruginosins 102A/B and 298A/B were also observed from other toxic strains of M. aeruginosa with positive fVIIa-sTF inhibitory activity. The active fractions contained cyanobacterial peptides of the aeruginosin class as fVIIa-sTF inhibitors detected by LC-MS.

Rivaroxaban delivery and reversal at a venous flow rate.

OBJECTIVE: Rivaroxaban is an oral anticoagulant that directly targets both free factor Xa and factor Xa in complex with its protein cofactor, factor Va, in the prothrombinase complex. It is approved in the United States for the prophylaxis of deep vein thrombosis and stroke in patients with atrial fibrillation; however, it also carries a black box warning regarding the risk of thrombosis after discontinuation of treatment. The purpose of this study was to determine the degree to which rivaroxaban, over a range of physiologically relevant free plasma concentrations, inhibits preassembled prothrombinase at a typical venous shear rate (100 s(-1)) and to determine the dynamics of rivaroxaban washout. METHODS AND RESULTS: Prothrombinase was assembled on phospholipid-coated glass capillaries. Its activity was characterized with respect to the activation of prothrombin (mean plasma concentration, 1.4 µmol/L) in the absence and presence of rivaroxaban (2, 5, and 10 nmol/L). The degree of inactivation of preassembled prothrombinase is sensitive to the solution-phase rivaroxaban concentration; however, prothrombinase unmasking upon removal of rivaroxaban is concentration independent. CONCLUSIONS: The model system presented suggests that when rivaroxaban plasma concentrations decrease after cessation of therapy, there will be an unmasking of thrombus-associated prothrombinase that may be related to the reported rebound phenomena.

Effect of telavancin (Vibativ) on routine coagulation test results.

Telavancin (Vibativ, Astellas Pharma US, Deerfield, IL) is a lipoglycopeptide antibiotic that has activity against gram-positive microorganisms, but also has the ability to bind to artificial phospholipids found in coagulation reagents. Normal pooled plasma was spiked with telavancin to obtain concentrations of 0, 12.5, 25, 50, 75, 100, 125, and 150 µg/mL of drug. Samples were tested using 3 different prothrombin time/international normalized ratio (INR) and activated partial thromboplastin time (aPTT) reagent systems, as well as for fibrinogen level, thrombin time, D-dimer level, dilute Russell viper venom time (DRVVT), protein C activity, and protein S activity. There was no effect of telavancin seen with non-phospholipid-dependent assays: fibrinogen level, thrombin time, and D-dimer testing. All INR and aPTT systems demonstrated concentration-dependent increases in clotting times, with Innovin (Siemens Healthcare Diagnostics, Deerfield, IL) INRs the most dramatic. False-positive DRVVT ratios started at 12.5 µg/mL of telavancin, with no effect on protein C or protein S levels until the telavancin level reached more than 100 µg/mL.

Anticoagulant effects of an antidiabetic drug on monocytes in vitro.

INTRODUCTION: Monocyte- and microparticle (MP)-associated tissue factor (TF) is upregulated in diabetes. Lipopolysaccharide (LPS) induces expression of TF and alternatively spliced TF (asTF) and increases MP release from monocytes. Using LPS-stimulated TF-bearing human monocytes, we examined whether glibenclamide, a sulfonylurea used to treat diabetes type 2, might possess anticoagulant properties. METHODS: We studied the effects of glibenclamide on cell- and supernatant-associated procoagulant activity (Factor Xa-generating assay and clot formation assay), on expression of TF and asTF (flow cytometry, RT-qPCR, western blot) and on cell viability and MP release (flow cytometry). RESULTS: Glibenclamide dose-dependently decreased procoagulant activity of cells and supernatants. The reduction in cellular procoagulant activity coincided with reduced expression of TF and asTF in cells, whereas cell viability remained almost unchanged. The glibenclamide-induced reduction in procoagulant activity of supernatants appeared to be associated with a decreased number of released MPs. CONCLUSIONS: Reduction of monocyte- and supernatant-associated procoagulant activity by glibenclamide is associated with decreased expression of TF and asTF and possibly with a reduced MP number. Our data indicate that glibenclamide reduces the prothrombotic state in LPS-stimulated monocytes in vitro. Glibenclamide might therefore also have an anticoagulant effect in vivo, but this needs to be further evaluated.

Effective treatment of chemoresistant breast cancer in vitro and in vivo by a factor VII-targeted photodynamic therapy.

BACKGROUND: The purpose of this study was to test a novel, dual tumour vascular endothelial cell (VEC)- and tumour cell-targeting factor VII-targeted Sn(IV) chlorin e6 photodynamic therapy (fVII-tPDT) by targeting a receptor tissue factor (TF) as an alternative treatment for chemoresistant breast cancer using a multidrug resistant (MDR) breast cancer line MCF-7/MDR. METHODS: The TF expression by the MCF-7/MDR breast cancer cells and tumour VECs in MCF-7/MDR tumours from mice was determined separately by flow cytometry and immunohistochemistry using anti-human or anti-murine TF antibodies. The efficacy of fVII-tPDT was tested in vitro and in vivo and was compared with non-targeted PDT for treatment of chemoresistant breast cancer. The in vitro efficacy was determined by a non-clonogenic assay using crystal violet staining for monolayers, and apoptosis and necrosis were assayed to elucidate the underlying mechanisms. The in vivo efficacy of fVII-tPDT was determined in a nude mouse model of subcutaneous MCF-7/MDR tumour xenograft by measuring tumour volume. RESULTS: To our knowledge, this is the first presentation showing that TF was expressed on tumour VECs in chemoresistant breast tumours from mice. The in vitro efficacy of fVII-tPDT was 12-fold stronger than that of ntPDT for MCF-7/MDR cancer cells, and the mechanism of action involved induction of apoptosis and necrosis. Moreover, fVII-tPDT was effective and safe for the treatment of chemoresistant breast tumours in the nude mouse model. CONCLUSIONS: We conclude that fVII-tPDT is effective and safe for the treatment of chemoresistant breast cancer, presumably by simultaneously targeting both the tumour neovasculature and chemoresistant cancer cells. Thus, this dual-targeting fVII-tPDT could also have therapeutic potential for the treatment of other chemoresistant cancers.

Challenges and approaches for assay development of membrane and membrane-associated proteins in drug discovery.

In addition to its role as a barrier between the cytoplasm and the extracellular milieu, the cell membrane is a scaffold for a diverse collection of receptors and enzymes. The organization afforded by this scaffold serves to ensure an efficient interaction between the components of the membrane. The desire to maintain this organization in solution is a challenge for the appropriate interrogation of these biochemical components. This chapter will discuss strategies that allow biochemical analysis of membrane-associated enzymes within standard biochemical reactions. The advantages of these screening strategies in identifying valuable compounds from compound libraries and in understanding the intricacies of complex multiprotein complexes (i.e., chemotaxis) will be discussed.

Weight-adjusted dalteparin for prevention of vascular thromboembolism in advanced pancreatic cancer patients decreases serum tissue factor and serum-mediated induction of cancer cell invasion.

The aim of the present study was to assess the role of tissue factor and serum-induced cell invasion in patients with advanced pancreatic cancer (APC). A cohort of 39 patients with APC, without thrombosis, receiving chemotherapy, were entered in a randomized controlled trial (ISRCTN = 76464767) of thromboprevention with weight-adjusted dalteparin (WAD). A total of 19 patients received WAD, the remaining 20 acting as a control group. Serum from baseline and week 8 was analysed for circulating-tissue factor antigen using ELISA. Circulating-tissue factor antigen rose from 324 pg/ml, [interquartile range (IQR) 282-347 pg/ml] to 356 pg/ml, (IQR 319-431 pg/ml) in controls (C), and decreased in the dalteparin-treated group (D) from 336 pg/ml (IQR 281-346 pg/ml) to 303 pg/ml (IQR 274-339 pg/ml). The difference in median percentage change between D and C was statistically significant [-4.0 (D) vs. 4.7 (C); P = 0.005, n = 39]. Serum-induced cellular invasion of MIA-Paca-2 cells in response to patient serum was studied using a Boyden chamber assay in 30 patients (14 WAD and 16 C). The median percentage change between C and D was significant [+54.9 (C) vs. -21.9 (D) P = 0.025, n = 30]. There was a weak correlation between BB-tissue factor reduction and cellular invasion reduction (Spearman) [0.384 (P = 0.037, n = 30)]. APC patients treated with WAD have lower tissue factor antigen levels and attenuated induction of cellular invasion in their blood. These assays may provide useful markers to guide appropriate dalteparin (and other low-molecular weight heparin) dosing schedules to optimize anticancer effects of dalteparin in APC.

Platelet tissue factor synthesis in type 2 diabetic patients is resistant to inhibition by insulin.

OBJECTIVE: Patients with type 2 diabetes have an increased risk of cardiovascular disease and show abnormalities in the coagulation cascade. We investigated whether increased synthesis of tissue factor (TF) by platelets could contribute to the hypercoagulant state. RESEARCH DESIGN AND METHODS: Platelets from type 2 diabetic patients and matched control subjects were adhered to different surface-coated proteins, and TF premRNA splicing, TF protein, and TF procoagulant activity were measured. RESULTS: Different adhesive proteins induced different levels of TF synthesis. A mimetic of active clopidogrel metabolite (AR-C69931 MX) reduced TF synthesis by 56 +/- 10%, an aspirin-like inhibitor (indomethacin) by 82 +/- 9%, and the combination by 96 +/- 2%, indicating that ADP release and thromboxane A(2) production followed by activation of P2Y12 and thromboxane receptors mediate surface-induced TF synthesis. Interference with intracellular pathways revealed inhibition by agents that raise cAMP and interfere with phosphatidylinositol 3-kinase/protein kinase B. Insulin is known to raise cAMP in platelets and inhibited collagen III-induced TF premRNA splicing and reduced TF activity by 35 +/- 5 and 47 +/- 5% at 1 and 100 nmol/l. Inhibition by insulin was reduced in type 2 diabetes platelets resulting in an approximately 1.6-fold higher TF synthesis than in matched control subjects. CONCLUSIONS: We characterized the extra- and intracellular mechanisms that couple surface activation to TF synthesis in adhering platelets. In healthy individuals, TF synthesis is inhibited by insulin, but in patients with type 2 diabetes inhibition is impaired. This leads to the novel finding that platelets from type 2 diabetic patients produce more TF than platelets from matched control subjects.

Paclitaxel potentiates inflammatory cytokine-induced prothrombotic molecules in endothelial cells.

To overcome the limitations of balloon expandible metal stent-induced neointimal smooth muscle cell proliferation, drug-coated stent devices have been developed. Drug eluting stents release high concentrations of antiproliferative agents, such as paclitaxel, to reduce neointimal hyperplasia. The proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), is known to cause severe endothelial dysfunction and accelerate atherosclerotic lesion progression. The interaction of TNF-alpha and paclitaxel on the release of prothrombotic molecules was examined in endothelial cells. Treatment of endothelial cells with paclitaxel had no direct effect on tissue factor (TF) expression, but TNF-alpha increased TF. Cotreatment of paclitaxel with TNF-alpha markedly augmented the release of TF. TNF-alpha induced release of plasminogen activator inhibitor but no synergism occurred with paclitaxel. Treatment of endothelial cells with paclitaxel and TNF-alpha reduced expression of thrombomodulin and protein C receptor. Tissue factor pathway inhibitor expression was reduced by prolonged treatment with either paclitaxel or TNF-alpha. The adhesion molecule, CD62 E, was induced by TNF-alpha; however, CD31, CD62 P, and CD106 were not affected by paclitaxel and TNF-alpha. Apoptosis was not observed with cotreatment of endothelial cells with paclitaxel and TNF-alpha. CD59-positive microparticles were released in response to TNF-alpha, but the release was not augmented by paclitaxel. Paclitaxel and TNF-alpha increased the nitrotyrosination of proteins. These findings indicate that paclitaxel enhances TNF-alpha-induced release of TF, and downregulated thrombomodulin, increased protein nitration, which may subsequently favor prothrombotic intimal surface.