Co-administration of nitric oxide-aspirin (NCX-4016) and aspirin prevents platelet and monocyte activation and protects against gastric damage induced by aspirin in humans

OBJECTIVES: The goal of this study was to test the hypothesis that NCX-4016 may have broader anti-inflammatory and antithrombotic effects as well as better gastric tolerability than aspirin in humans. BACKGROUND: NCX-4016 is an aspirin derivative containing a nitric oxide-releasing moiety that prevents platelet activation and modulates tissue factor (TF) expression and cytokine release from lipopolysaccharide (LPS)-stimulated monocytes. METHODS: This was a blind-observer, placebo-controlled, parallel-group study in which 48 healthy subjects were randomized to receive NCX-4016 800 mg twice a day, NCX-4016 800 mg twice a day plus aspirin 325 mg, aspirin 325 mg, or placebo for 21 days. RESULTS: Similar to aspirin alone, NCX-4016 effectively inhibited platelet aggregation induced by 0.6 mmol/ arachidonic acid, clot-stimulated thromboxane (TX) B2 generation in whole blood, and urinary excretion of 11-dehydro-TXB2. Unlike aspirin alone, the administration of NCX-4016 significantly inhibited TF expression in monocytes stimulated ex vivo with 10 micromol/l LPS (determined by flow-cytometry analysis of TF on CD14 positive cells). NCX-4016 also inhibited the rapid TF expression induced in monocytes by a proteinase activated receptor agonist (thrombin receptor activator protein, 2 micromol/l) as well as LPS-induced expression of CD11b . Ex vivo, release of MCP-1 and interleukin-6 were significantly inhibited by NCX-4016, but not by aspirin. NCX-4016 was not associated with gastric damage, and significantly reduced gastric injury when co-administered with aspirin, although both drugs reduced gastric PGE2 production to the same extent. CONCLUSIONS: NCX-4016 is equally effective as aspirin in inhibiting cyclooxygenase activity. However, NCX-4016 causes less gastric damage and prevents monocyte activation. Larger multicenter trials are warranted to establish clinical efficacy and safety of NCX-4016.     

Vitamin E reduces monocyte tissue factor expression in cirrhotic patients

Clotting activation may occur in liver cirrhosis, but the pathophysiological mechanism has not been fully elucidated. Because a previous study demonstrated that lipid peroxidation is increased in cirrhosis, we analyzed whether there is a relationship between lipid peroxidation and clotting activation. Thirty cirrhotic patients (19 men and 11 women; age, 34 to 79 years) and 30 controls matched for sex and age were investigated. In all subjects, monocyte expression of tissue factor (TF) antigen and activity; plasma levels of prothrombin fragment 1+2 (F1+2), a marker of thrombin generation; and urinary excretion of Isoprostane-F2alpha-III, a marker of lipid peroxidation, were measured. Furthermore, the above-reported variables were re-evaluated after 30 days of treatment with standard therapy (n = 5) or standard therapy plus 300 mg vitamin E twice daily (n = 9). In addition, we analyzed in vitro if vitamin E (50 micromol/L) influenced monocyte TF expression and F1+2 generation. Cirrhotic patients had higher values of Isoprostane-F2alpha-III (P <. 0001), F1+2 (P <.0001), and monocyte TF antigen (P <.0001) and activity (P <.03) than controls. Isoprostane-F2alpha-III was significantly correlated with F1+2 (Rho = 0.85; P <.0001) and TF antigen (Rho = 0.95; P <.0001) and activity (Rho = 0.94; P <.0001). After vitamin E treatment, Isoprostane-F2alpha-III (P =.008), F1+2 (P <.008), and monocyte TF antigen (P =.012) and activity (P =.008) significantly decreased; no changes of these variables were detected in patients not receiving vitamin E. In vitro, vitamin E significantly reduced the expression of monocyte TF antigen (-52%; P =.001) and activity (-55%; P =.003), as well as F1+2 generation (-51%; P =.025). This study shows that vitamin E reduces both lipid peroxidation and clotting activation and suggests that lipid peroxidation may be an important mediator of clotting activation in liver cirrhosis.     

Enhanced monocyte tissue factor expression in hepatosplenic schistosomiasis.

Monocyte tissue factor expression was evaluated in 67 patients with hepatosplenic Schistosomiasis. They were classified as Child A (n = 15), Child B (n = 15), Child C (n = 12) and Bleeders (n = 10), in addition to 15 healthy controls. Mononuclear cells were cultured in vitro with and without lipopolysaccharide (LPS) to assess monocyte tissue factor (TF) antigen (Ag) and activity (Act) in cell lysate, in addition to measurement of prothrombin fragment 1 + 2 (F1 + 2) as a marker of in vivo thrombin generation. A significant increase in monocyte TF Ag and TF Act was noted in all stages of the disease compared with the control group, with marked accentuation during an acute attack of variceal bleeding. This enhanced monocyte expression was noted before the addition of LPS and became more obvious with addition of LPS. An increasing level of F1 + 2 was similarly noted. These findings constitute further evidence for an existing prothrombotic state in hepatosplenic Schistosomiasis, and also that monocytes are closely implicated in the haemostatic diathesis characterizing the disease.     

The in vivo kinetics of tissue factor messenger RNA expression during human endotoxemia: relationship with activation of coagulation

Triggering of the tissue factor (TF)-dependent coagulation pathway is considered to underlie the generation of a procoagulant state during endotoxemia. To determine the in vivo pattern of monocytic TF messenger RNA (mRNA) expression during endotoxemia, 10 healthy volunteers were injected with lipopolysaccharide (LPS, 4 ng/kg) and blood was collected before and 0.5, 1, 2, 3, 4, 6, 8, and 24 hours after LPS administration. Total blood RNA was isolated and amplified by NASBA (nucleic acid sequence-based amplification), followed by quantitation of TF mRNA by an electrochemiluminescence (ECL) assay. To compare the pattern of coagulation activation with the kinetics of monocytic TF mRNA expression, we measured plasma levels of markers of thrombin generation, thrombin-antithrombin (TAT) complexes, and prothrombin fragment 1 + 2 (F1 + 2). Baseline value (mean +/- SEM) of the number of TF mRNA molecules per monocytic cell was 0.08 +/- 0.02. A progressive and significant (P <.0001) increase in TF expression was observed after LPS injection (+0.5 hour: 0.3 +/- 0.1, +1 hour: 1.3 +/- 0.9, +2 hours: 4.1 +/- 0.9), peaking at +3 hours (10 +/- 1.9 TF mRNA molecules per monocyte). As TF mRNA levels increased, thrombin generation was augmented. Peak levels of TAT and F1 + 2 were reached later (at t +4 hours) than those of TF mRNA. TF mRNA, TAT, and F1 + 2 levels returned to baseline after 24 hours. In conclusion, we used a NASBA/ECL-based technique to quantify TF mRNA in whole blood during human endotoxemia and observed a 125-fold increase in TF mRNA levels. Our data demonstrate a pivotal role for enhanced TF gene activity in the activation of coagulation after LPS challenge. (Blood. 2000;96:554-559)     

CD40 ligand enhances monocyte tissue factor expression and thrombin generation via oxidative stress in patients with hypercholesterolemia

OBJECTIVES: We tested the hypothesis that CD40 ligand (CD40L) induces a prothrombotic state by enhancing oxidative stress. BACKGROUND: Patients with hypercholesterolemia show an ongoing prothrombotic state, but the underlying mechanism is still unclear. METHODS: Circulating levels of the soluble form of CD40L (sCD40L), prothrombin fragment (F1+2, a marker of thrombin generation), and 8-hydroxy-2'-deoxyguanosine (8-OHdG, a marker of oxidative stress) were measured in 40 patients with hypercholesterolemia and in 20 age- and gender-matched healthy subjects. RESULTS: Patients with hypercholesterolemia showed significantly higher levels of sCD40L (p <0.005), 8-OHdG (p <0.005), and prothrombin F1+2 (p <0.005), as compared with control subjects. Soluble CD40L significantly correlated with 8-OHdG (r=0.85, p <0.0001) and prothrombin F1+2 (r=0.83, p <0.0001); a significant correlation between 8-OHdG and prothrombin F1+2 was also observed (r=0.64, p <0.0001). An in vitro study demonstrated that CD40L-stimulated monocytes from patients with hypercholesterolemia expressed more tissue factor (TF) and prothrombin F1+2 than monocytes from controls; co-incubation of monocytes with either an inhibitor of NADPH oxidase or an inhibitor of phosphatidylinositol-3-kinase significantly reduced CD40L-mediated clotting activation. A marked inhibition of CD40L-mediated clotting activation was also observed in two male patients with hereditary deficiency of gp91 phox, the central core of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Finally, we demonstrated that CD40L-mediated clotting activation was significantly inhibited by vitamin C, a known antioxidant. CONCLUSIONS: This study indicates that in patients with hypercholesterolemia, CD40L over-expresses TF and increases the thrombin generation rate by an oxidative stress-mediated mechanism that requires the activation of NADPH oxidase.     

Heparin blunts endotoxin-induced coagulation activation

BACKGROUND: Lipopolysaccharide (LPS) is a major trigger of sepsis-induced disseminated intravascular coagulation (DIC) via the tissue factor (TF)/factor VIIa-dependent pathway of coagulation. Experimental endotoxemia has been used repeatedly to explore this complex pathophysiology, but little is known about the effects of clinically used anticoagulants in this setting. Therefore, we compared with placebo the effects of unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) on LPS-induced coagulation. METHODS AND RESULTS: In a randomized, double-blind, placebo-controlled trial, 30 healthy male volunteers received LPS 2 ng/kg IV followed by a bolus-primed continuous infusion of UFH, LMWH, or placebo. In the placebo group, activation of coagulation caused marked increases in plasma levels of prothrombin fragment F(1+2) (P<0.01) and polymerized soluble fibrin, termed thrombus precursor protein (TpP; P<0.01); TF-positive monocytes doubled in response to LPS, whereas levels of activated factor VII slightly decreased and levels of TF pathway inhibitor remained unchanged. UFH and LMWH markedly decreased activation of coagulation caused by LPS, as F(1+2) and TpP levels only slightly increased; TF expression on monocytes was also markedly reduced by UFH. TF pathway inhibitor values increased after either heparin infusion (P<0.01). Concomitantly, factor VIIa levels dropped by >50% at 50 minutes after initiation of either heparin infusion (P<0.01). CONCLUSIONS: This experimental model proved the anticoagulatory potency of UFH and LMWH in the initial phase of experimental LPS-induced coagulation. Successful inhibition of thrombin generation also translates into blunted activation of coagulation factors upstream and downstream of thrombin.     

Endotoxin-induced activation of the coagulation cascade in humans: effect of acetylsalicylic acid and acetaminophen.

During Gram-negative septic shock, lipopolysaccharide (LPS, endotoxin) induces tissue factor (TF) expression. TF expression is mediated by nuclear factor kappaB and amplified by activated platelets. TF forms a highly procoagulant complex with activated coagulation factor VII (FVIIa). Hence, we hypothesized that aspirin, which inhibits LPS-induced, nuclear factor kappaB-dependent TF expression in vitro and platelet activation in vivo, may suppress LPS-induced coagulation in humans. Therefore, we studied the effects of aspirin on systemic coagulation activation in the established and controlled setting of the human LPS model. Thirty healthy volunteers were challenged with LPS (4 ng/kg IV) after intake of either placebo or aspirin (1000 mg). Acetaminophen (1000 mg) was given to a third group to control for potential effects of antipyresis. Neither aspirin nor acetaminophen inhibited LPS-induced coagulation. However, LPS increased the percentage of circulating TF(+) monocytes by 2-fold. This increase was associated with a decrease in FVIIa levels, which reached a minimum of 50% 24 hours after LPS infusion. Furthermore, LPS-induced thrombin generation increased plasma levels of circulating polymerized, but not cross-linked, fibrin (ie, thrombus precursor protein), whereas levels of soluble fibrin were unaffected. In summary, a single 1000-mg dose of aspirin did not decrease LPS-induced coagulation. However, our study showed, for the first time, that LPS increases TF(+) monocytes, substantially decreases FVIIa levels, and enhances plasma levels of thrombus precursor protein, which may be a useful marker of fibrin formation in humans.     

Monocytes, but not endothelial cells, downregulate the anticoagulant activity of activated protein C

Activated protein C (APC) is a natural anticoagulant and inhibits thrombin generation by degrading factors Va and VIIIa. We evaluated the ability of APC to inhibit blood coagulation triggered by lipopolysaccharide (LPS)-stimulated [tissue factor (TF)-expressing] human mononuclear cells (MNCs) or umbilical vein endothelial cells (HUVECs). Using a plasma recalcification assay, we found that APC (up to 53.3 nmol/l final concentration) had a poor anticoagulant effect in the presence of LPS-stimulated MNCs, whereas it caused a marked prolongation of clotting time in the presence of LPS-stimulated HUVECs. A poor response to APC was also observed when platelet-free MNCs, monocyte-enriched preparations or the monocytoid cell line U937 were tested. Using a TF-independent (FXa-induced) thrombin generation assay, we demonstrated that both LPS-stimulated and unstimulated MNCs negated the inhibitory activity of APC. Direct determination of FVa activity indicated that MNCs were less efficient than HUVECs in promoting FVa inactivation by APC. Together, our results suggest that MNCs, at variance with HUVECs, protect factor Va from inactivation by APC, probably through the expression of a membrane component not present on endothelial cells. These strengthen the importance of monocytes in fibrin deposition associated with pathological conditions characterized by monocyte recruitment and activation.     

Monocyte tissue factor induction by lipopolysaccharide (LPS): dependence on LPS-binding protein and CD14, and inhibition by a recombinant fragment of bactericidal/permeability-increasing protein

Mononuclear phagocytes, stimulated by bacterial lipopolysaccharide (LPS), have been implicated in the activation of coagulation in sepsis and endotoxemia. In monocytes LPS induces the synthesis of tissue factor (TF) which, assembled with factor VII, initiates the blood coagulation cascades. In this study we investigated the mechanism of LPS recognition by monocytes, and the consequent expression of TF mRNA and TF activity. We also studied the inhibition of these effects of LPS by rBPI23, a 23-kD recombinant fragment of bactericidal/permeability increasing protein, which has been shown to antagonize LPS in vitro and in vivo. Human peripheral blood mononuclear cells, or monocytes isolated by adherence, were stimulated with Escherichia coli O113 LPS at physiologically relevant concentrations (> or = 10 pg/mL). The effect of LPS was dependent on the presence of the serum protein LBP (lipopolysaccharide-binding protein), as shown by the potentiating effect of human recombinant LBP or serum. Furthermore, recognition of low amounts of LPS by monocytes was also dependent on CD14 receptors, because monoclonal antibodies against CD14 greatly reduced the LPS sensitivity of monocytes in the presence of serum or rLBP. Induction of TF activity and mRNA expression by LPS were inhibited by rBPI23. The expression of tumor necrosis factor showed qualitatively similar changes. Considering the involvement of LPS-induced TF in the potentially lethal intravascular coagulation in sepsis, inhibition of TF induction by rBPI23 may be of therapeutic benefit.     

Enhanced expression of monocyte tissue factor in patients with liver cirrhosis

Background—Previous studies have shown that cirrhotic patients produce increased amounts of thrombin but the underlying mechanism is still unknown.
Aims—To analyse the relation between the rate of thrombin generation and monocyte expression of tissue factor (TF) in cirrhosis.
Patients—Thirty three cirrhotic patients classified as having low (n=7), moderate (n=17), or severe (n=9) liver failure according to Child-Pugh criteria.
Methods—Prothrombin fragment F1+2, monocyte TF activity and antigen, and endotoxaemia were measured in all patients. Polymerase chain reaction (PCR) analysis of TF mRNA was performed in monocytes of five cirrhotic patients.
Results—Prothrombin fragment F1+2 was higher in cirrhotic patients than in controls (p<0.0001). Monocytes from cirrhotic patients had higher TF activity and antigen than those from controls (p<0.001) with a progressive increase from low to severe liver failure. Monocyte expression of TF was significantly correlated with plasma levels of F1+2 (TF activity: r=0.98, p<0.0001; TF antigen: r=0.95, p<0.0001) and with endotoxaemia (TF activity: r=0.94, p<0.0001; TF antigen: r=0.91, p<0.0001). PCR analysis of TF mRNA showed TF expression only in three patients with endotoxaemia (more than 15 pg/ml).
Conclusions—Cirrhotic patients have enhanced expression of TF which could be responsible for clotting activation, suggesting that endotoxaemia might play a pivotal role.

     

Increase in expression of monocytic tissue factor (CD142) with monocytes and blood platelet activation in liver cirrhosis.

Tissue factor (TF) is one of the proteins that participate in hemostatic and inflammatory processes. Activated monocytes present in the liver increase expression of TF, and while accumulating in the organ they can intensify inflammation. The aim of the present study was to evaluate the expression of TF on monocytes in advanced liver cirrhosis with regard to other activation markers. The flow cytometric analysis of TF (CD142), CD14, adhesive molecules CD11b and CD11c, costimulatory molecules CD40, CD80 and CD86, and HLA-DR on monocytes was carried out in 45 patients with postalcoholic liver cirrhosis (Child Pugh B, 20 patients; Child Pugh C, 25 patients) and in 25 healthy persons. The positive correlation between monocytic TF expression and monocyte [soluble CD14 (sCD14), CD11b, monocyte aggregates] activation, the expression of costimulatory molecules on monocytes (CD40, CD80), blood platelet (soluble P-selectin, microplatelets) activation, the level of tumor necrosis factor-alpha, biochemical parameters of liver damage (alanine aminotransferase, aspartate aminotransferase, alkaline phosphate, gamma-glutamyltransferase, and bilirubin) as well as coagulation disorders were observed in the study. In conclusion, the study revealed that the activation of monocytes and blood platelets is accompanied by the elevation of monocytic TF expression in advanced liver cirrhosis. The monocytic TF is a significant link connecting clotting processes and inflammatory and immunological phenomena in liver cirrhosis.     

Importance of C-reactive protein in regulating monocyte tissue factor expression in patients with inflammatory rheumatic diseases

OBJECTIVE: To determine the relationship between plasma C-reactive protein (CRP) concentrations and monocyte tissue factor (TF) expression induced in vitro by combinations of CRP, ss2-glycoprotein I (ss2-GPI), and lipopolysaccharide (LPS). METHODS: Peripheral blood mononuclear cells (PBMC) from 26 healthy individuals and 31 patients with inflammatory rheumatic diseases (IRD) were cultured with combinations of CRP, purified or recombinant ss2-GPI, and LPS and monocyte TF procoagulant activity, TF antigen, and TF mRNA were measured. Results were examined against plasma CRP levels. RESULTS: Monocytes from patients with IRD expressed significantly more TF when stimulated with CRP compared to normal monocytes (p = 0.002). An incremental positive correlation was observed between plasma CRP levels and TF induced by CRP or ss2-GPI. Significantly more TF was induced with CRP combined with ss2-GPI, compared to ss2-GPI alone, either with costimulation or CRP priming. Conversely, when combined with LPS, ss2-GPI suppressed TF induction in a dose-dependent manner on normal PBMC but not on PBMC from patients with IRD. The loss of suppression correlated strongly with plasma CRP levels. CONCLUSION: This study shows a remarkably consistent effect of CRP on monocyte TF expression. Systemic inflammation associated with elevated plasma CRP conferred a phenotype on PBMC, whereby incremental priming with respect to TF expression (induced by CRP itself or ss2-GPI) was apparent, and ss2-GPI-mediated inhibition of TF expression induced by LPS was incrementally lost. CRP regulation of monocyte TF could contribute to the higher than expected atherosclerotic vascular disease seen in patients with IRD.     

Induction of tissue factor expression in human monocyte/endothelium cocultures

Summary. Induction of tissue factor (TF) expression on monocyctes and endothelial cells is central to the development of septic coagulopathy. Serum concentrations of endotoxin in septic patients who develop disseminated intravascular coagulation (DIC) do not, however, reach the levels that would directly stimulate TF expression on either monocytes or endothelium. We show, using an in vitro coculture system, that the interaction of monocytes with endothelium induces the expression of significant levels of TF. Unstimulated cocultures of monocytes (2 × 10⁴/well) and endothelial cells (2 × 10⁴/well) produced 35.3± 8.5 mU of PCA/well, representing a 5-fold increase over the combined PCA of each cell type cultured alone (7.1 ± 1.5 mU, n = 6, P < 0.001). Significant enhancement was also found in the presence of low concentrations of LPS. Induction of TF protein was confirmed by Western blotting. Fixation of monocytes with paraformaldehyde completely abolished TF induction in cocultures, whereas fixation of endothelium had no effect, suggesting that TF induction occurred in monocytes rather than endothelial cells. Induction of TF in cocultures could be further augmented by preincubating the endothelial cells with IFN-γ. When endothelium was prestimulated with 500U/ml IFN-γ there was 142 ±11% increase over unstimulated cocultures (n = 5, P< 0.01). TF induction was inhibited by 32 ± 6% in the presence of anti-ICAM-1 mAb (n = 5, P < 001). Our results suggest that monocyte interactions with vascular endothelium, regulated by inflammatory cytokines, and mediated by adhesive ligand binding, leads to the induction of functional monocyte TF protein, which may be responsible for the initiation of DIC in sepsis.     

Interleukin 10 regulates cellular responses in monocyte/endothelial cell co-cultures

Adhesive interactions between monocytes and vascular endothelial cells increase the expression of the inflammatory genes, tissue factor (TF) and E-selectin, thus contributing to the inflammatory process. In this study, we have shown that these responses could be regulated by the immunomodulatory cytokine interleukin 10 (IL-10). IL-10 reduced TF generation in monocyte/endothelium co-cultures (64. 3 +/- 3.3% reduction, P < 0.01, n = 4) by acting directly on monocytes, whereas IL-4 inhibited TF expression in both monocytes and endothelium. Similarly, IL-10 reduced the induction of endothelial E-selectin by monocytes (100% reduction at 21 h), but had no effect on cytokine-induced E-selectin expression. IL-10 itself was not able to induce E-selectin protein or mRNA in endothelial cells. IL-10 mRNA was detected in monocytes after 6 h co-culture with endothelial cells, and was sustained for up to 30 h. Finally, IL-10 significantly reduced the adhesion of monocytes to endothelium (45% reduction), which may account in part for the inhibitory actions of IL-10. We conclude that IL-10 has an anti-inflammatory effect on monocyte/endothelium interactions, and may itself be produced as a result of such interactions.     

Plasma tissue factor plus activated peripheral mononuclear cells activate factors VII and X in cardiac surgical wounds

OBJECTIVES: The purpose of this study was to test the hypothesis that activated monocytes with soluble plasma tissue factor (pTF) activate factors VII and X to generate thrombin. BACKGROUND: Despite heparin, thrombin is progressively generated during cardiac surgery with cardiopulmonary bypass (CPB), produces intravascular fibrin and fibrinolysis, and causes serious thromboembolic and nonsurgical bleeding complications. Thrombin is primarily produced in the surgical wound, but mechanisms are unclear. METHODS: In 13 patients, interactions of mononuclear cells, platelets, pTF, and pTF fractions to activate factors VII and X were evaluated in pre-bypass, perfusate, and pericardial wound blood before and during CPB. RESULTS: Monocytes are activated in wound, but not in pre-bypass or perfusate plasma (monocyte chemotactic protein-1 = 29.5 +/- 2.1 pmoles/l vs. 2.8 +/- 1.2 pmoles/l and 3.3 +/-1.4 pmoles/l, respectively). Wound pTF is substantially elevated compared to other locations (3.64 +/- 0.45 pmoles/l vs. 0.71 +/- 0.65 pmoles/l and 1.31 +/- 1.4 pmoles/l). Supernatant wound pTF contains 81.7% of TF antigen; wound microparticle pTF contains 18.3%. Wound monocytes and all C5a-stimulated monocytes (but not activated platelets) completely convert factor VII to factor VIIa with wound pTF. Activated monocytes more efficiently activate factor X with wound supernatant TF/factor VII(VIIa) complex than with wound microparticle TF/factor VII(fVIIa). The correlation coefficient (r) between wound thrombin generation (F1.2) and wound pTF concentration is 0.944 (p = 0.0004). CONCLUSIONS: During cardiac surgery with CPB, wound monocytes plus wound pTF or wound microparticle-free supernatant pTF preferentially accelerate activation of factor VII and factor X. This system represents a novel mechanism for thrombin generation via the TF coagulation pathway.     

Anisodamine counteracts lipopolysaccharide-induced tissue factor and plasminogen activator inhibitor-1 expression in human endothelial cells: contribution of the NF-kappa b pathway

In this study we aimed to investigate whether the therapeutic efficacy of anisodamine in the treatment of bacteraemic shock could--at least in part--be brought about by its direct interference with the lipopolysaccharide (LPS)-induced activation of endothelial cells. Thus, we investigated the effect of anisodamine on LPS-induced expression of plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF), two major markers of endothelial activation. PAI-1 was measured in the conditioned media of human umbilical vein endothelial cells (HUVEC) by a specific enzyme-linked immunosorbent assay (ELISA) whereas TF activity was measured in the lysates of these cells by using a single step clotting assay. Results obtained in these assays were confirmed on the level of specific mRNA expression by Northern blotting using specific probes for human PAI-1 or TF. In order to evaluate a possible contribution of the NF-kappa B pathway on the effects observed, electrophoretic mobility shift assays (EMSA) were performed using nuclear extracts from HUVEC and NF-kappa B-binding oligonucleotides. When HUVEC were treated with 1 microg/ml LPS a significant increase in PAI-1 and TF activity was observed compared with cells incubated without LPS. Anisodamine dose-dependently inhibited this LPS-induced upregulation of PAI-1 and TF. Anisodamine alone had no effect on the constitutive expression of PAI-1 and TF in these cells. These effects were also confirmed on the level of specific PAI-1 and TF mRNA expression by Northern blotting. Furthermore, we could show by EMSA that anisodamine completely abolished LPS-induced NF-kappa B DNA binding activity in nuclear extracts from HUVEC treated with LPS together with anisodamine. Thus, we provide evidence that anisodamine counteracts endothelial cell activation by inhibiting LPS-induced PAI-1 and TF expression in these cells. Its interference with the NF-kappa B pathway might - at least in part - contribute to this effect. The ability of anisodamine to counteract LPS effects on endothelial cells might be one underlying mechanism explaining its efficacy in the treatment of bacteraemic shock.     

Platelet-CD40 ligand interaction with melanoma cell and monocyte CD40 enhances cellular procoagulant activity.

Platelet-tumor cell interactions are believed to be important in tumor metastasis. Tumor cell tissue factor (TF) expression enhances metastasis and angiogenesis, and is primarily responsible for tumor-induced thrombin generation and the formation of tumor cell-platelet aggregates. Activated platelets express and release CD40 ligand (CD40L), which induces endothelial TF expression by ligation to CD40. We investigated the effect of platelet-derived CD40L on the TF activity of human CD40-positive melanoma cells and monocytes by incubating supernatants from activated or resting platelets with tumor cells or monocytes, and by bringing resting or activated platelets into close apposition with tumor cell monolayers. CD40L was present on the surface of activated (but not resting) platelets and was also released following platelet activation. Both recombinant soluble CD40L (rsCD40L) and activated platelet supernatants increased procoagulant activity (PCA) and TF antigen in tumor cells and monocytes. The increase in TF activity induced by both rsCD40L and activated platelet supernatants was inhibited by anti-CD40L antibody. Furthermore, contact of activated platelets with tumor cells increased cellular PCA, and this effect was also inhibited by anti-CD40L. In malignancy, the increase in cellular TF activity via CD40 (tumor cell)-CD40L (platelet) interaction may possibly enhance intravascular coagulation and hematogenous metastasis.