Comparison of assays measuring extracellular vesicle tissue factor in plasma samples: communication from the ISTH SSC Subcommittee on Vascular Biology.

BACKGROUND: Scientific and clinical interest in extracellular vesicles (EVs) is growing. EVs that expose tissue factor (TF) bind factor VII/VIIa and can trigger coagulation. Highly procoagulant TF-exposing EVs are detectable in the circulation in various diseases, such as sepsis, COVID-19, or cancer. Many in-house and commercially available assays have been developed to measure EV-TF activity and antigen, but only a few studies have compared some of these assays. OBJECTIVES: The International Society on Thrombosis and Haemostasis Scientific and Standardization Committee Subcommittee on Vascular Biology initiated a multicenter study to compare the sensitivity, specificity, and reproducibility of these assays. METHODS: Platelet-depleted plasma samples were prepared from blood of healthy donors. The plasma samples were spiked either with EVs from human milk or EVs from TF-positive and TF-negative cell lines. Plasma was also prepared from whole human blood with or without lipopolysaccharide stimulation. Twenty-one laboratories measured EV-TF activity and antigen in the prepared samples using their own assays representing 18 functional and 9 antigenic assays. RESULTS: There was a large variability in the absolute values for the different EV-TF activity and antigen assays. Activity assays had higher specificity and sensitivity compared with antigen assays. In addition, there was a large intra-assay and interassay variability. Functional assays that used a blocking anti-TF antibody or immunocapture were the most specific and sensitive. Activity assays that used immunocapture had a lower coefficient of variation compared with assays that isolated EVs by high-speed centrifugation. CONCLUSION: Based on this multicenter study, we recommend measuring EV-TF using a functional assay in the presence of an anti-TF antibody.

Cold-pressed minke whale oil reduces circulating LDL/VLDL-cholesterol, lipid oxidation and atherogenesis in apolipoprotein E-deficient mice fed a Western-type diet for 13 weeks.

BACKGROUND: Long-chain n3-polyunsaturated fatty acids (LC n3-PUFA) are well known for their anti-inflammatory activity and their impact on cardiovascular disease. Cold-pressed whale oil (CWO) has half the amount of LC n3-PUFA compared to cod liver oil (CLO). Still, there has been observed more pronounced beneficial effects on cardiovascular disease markers from intake of CWO compared to intake of CLO in human intervention studies. Extracts from CWO deprived of fatty acids have also been shown to display antioxidative and anti-inflammatory effects in vitro. The aim of this study was to investigate whether intake of a high-fat Western-type diet (WD) supplemented with CWO would prevent the development of atherosclerotic lesions in apolipoprotein E-deficient (ApoE-/-) mice. METHODS: Seventy female ApoE-/- mice were fed a WD containing 1% CWO, CLO or corn oil (CO). Atherosclerotic lesion formation, body and tissue weights, hepatic gene expression together with serum levels of LDL/VLDL-cholesterol, ox-LDL, total antioxidant status and various serum cardiovascular disease/proinflammatory markers were evaluated. Statistical analyses were performed using SPSS, and Shapiro-Wilk's test was performed to determine the distribution of the variables. Statistical difference was assessed using One-Way ANOVA with Tukeys' post hoc test or Kruskal-Wallis test. The hepatic relative gene expression was analysed with REST 2009 (V2.0.13). RESULTS: Mice fed CWO had less atherosclerotic lesions in the aortic arch compared to mice fed CO. Levels of LDL/VLDL-cholesterol and ox-LDL-cholesterol were also markedly reduced whereas total antioxidant levels were enhanced in mice fed CWO compared to CO-fed mice. In addition, CWO-fed mice gained less weight and several hepatic genes involved in the cholesterol metabolism were up-regulated compared to CO-fed mice. CONCLUSION: In the present study mice fed a WD supplemented with 1% CWO had reduced formation of atherosclerotic lesions in the aortic arch, reduced serum LDL/VLDL-cholesterol and ox-LDL-cholesterol, increased serum total antioxidant status and reduced body weight compared to mice fed a WD supplemented with 1% CO.

Antioxidant and Anti-Inflammatory Activities in Extracts from Minke Whale (Balaenoptera acutorostrata) Blubber.

Intake of long-chain omega-3 polyunsaturated fatty acids (LC-n3-PUFA) is commonly recognized to reduce cardiovascular disease (CVD). In previous studies, cold-pressed whale oil (CWO) and cod liver oil (CLO) were given as a dietary supplement to healthy volunteers. Even though CWO contains less than half the amount of LC-n3-PUFA of CLO, CWO supplement resulted in beneficial effects on anti-inflammatory and CVD risk markers compared to CLO. In the present study, we prepared virtually lipid-free extracts from CWO and CLO and evaluated the antioxidative capacity (AOC) and anti-inflammatory effects. Oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays were used to test the AOC, and the results indicated high levels of antioxidants present in all extracts. The anti-inflammatory effects of the extracts were tested with lipopolysaccharide- (LPS-) treated THP-1 cells, measuring its ability to reduce cytokine and chemokine secretion. Several CWO extracts displayed anti-inflammatory activity, and a butyl alcohol extract of CWO most effectively reduced TNF-α (50%, p < 0.05) and MCP-1 (85%, p < 0.001) secretion. This extract maintained a stable effect of reducing MCP-1 secretion (60%, p < 0.05) even after long-term storage. In conclusion, CWO has antioxidant and anti-inflammatory activities that may act in addition to its well-known LC-n3-PUFA effects.

Laboratory characterization of leukemic cell procoagulants.

BACKGROUND: In acute myeloid leukemias, there is an increased chance to develop thrombotic disorders. We hypothesized that in addition to leukemic promyelocytes, monocytic leukemia cells may also have a higher procoagulant activity. METHODS: Fibrin formation was assessed by a one-stage clotting assay using a magnetic coagulometer. The thrombin generation test (TGT) of magnetically isolated normal human monocytes, intact leukemic cells and their isolated microparticles was performed by a fluorimetric assay. Phosphatidylserine (PS) expression of leukemic cells and microparticle number determinations were carried out by flow cytometry. RESULTS: All cell lines displayed a significant procoagulant potential compared to isolated normal human monocytes. In the TGT test, the mean of lagtime and the time to peak parameters were significantly shorter in leukemic cells (3.9-4.7 and 9.9-10.3 min) compared to monocytes (14.9 and 26.5 min). The mean of peak thrombin in various monocytic leukemia cell lines was 112.1-132.9 nM vs. 75.1 nM in monocytes; however, no significant difference was observed in the ETP parameter. Factor VII-deficient plasma abolished all procoagulant activity, whereas factor XII-deficient plasma did not affect the speed of fibrin formation and thrombin generation but modulated the amount of thrombin. Factor XI-deficient plasma affected the time to peak values in one leukemic cell line and also attenuated peak thrombin. Leukemia cell-derived microparticles from all three cell lines exerted a procoagulant effect by significantly shortening the lagtime in TGT; there was a nonsignificant difference in case of ETP parameter. CONCLUSIONS: All investigated monocytic leukemia cell lines exhibited significant thrombin generation. This phenomenon was achieved by the procoagulants on the surface of leukemic cells as well as by their microparticles.

The immunomodulatory effects of barettin and involvement of the kinases CAMK1α and RIPK2.

BACKGROUND: Barettin is a marine natural compound with reported anti-inflammatory and antioxidant properties. The combination of these effects led us to explore barettin further as an inhibitor of atherosclerosis development. METHODS: The effect of barettin on MCP-1 and IL-10 secretion from activated immune cells was detected by ELISA. Determination of cell viability of oxidized low-density lipoprotein (oxLDL) and barettin exposed HUVEC cells were investigated by using CellTiter 96® AQ(ueous) One Solution. The kinase inhibition assays were performed using a radioactive ((33)P-ATP) filter binding assay at the University of Dundee, UK. RESULTS: Barettin reduces the secretion of monocyte chemotactic protein-1 (MCP-1) from LPS-stimulated monocytes, but was not able to prevent oxLDL-induced cell death in HUVEC. Barettin has inhibitory activity against two protein kinases related to inflammation, namely the receptor-interacting serine/threonine kinase 2 (RIPK2) and calcium/calmodulin-dependent protein kinase 1α (CAMK1α). We also demonstrate that barettin reduce the production of the anti-inflammatory cytokine interleukin-10 (IL-10) in a dose and time-dependent manner, possibly by inhibiting CAMK1α. CONCLUSIONS: The anti-inflammatory activity of barettin is exerted through the regulation of inflammatory mediators such as MCP-1 and IL-10, possibly via inhibition of kinases.

Pro- and anti-inflammatory effects of histamine on tissue factor and TNFα expression in monocytes of human blood.

BACKGROUND: Histamine is classified as an inflammatory mediator and has been reported to have anti- as well as pro-inflammatory properties. The aim of this study was to explore the role of histamine on the production of LPS-induced tissue factor (TF) activity and TNFα in monocytes of whole blood in the absence and presence of TNFα or PMA. METHODS: Human blood anticoagulated with Fragmin was subjected to stimulation by LPS in the presence and absence of TNFα or PMA and various concentrations of histamine. Tissue factor (TF) activity was measured in lyzed cells after isolation of mononuclear cells whereas TNFα was quantified in plasma after centrifugation of cells. RESULTS: Histamine gave a dose dependent inhibitory effect on LPS-induced TF activity in monocytes of whole blood, with a 50% reduction at 0.033 µM. A similar effect was seen when the blood cells were stimulated with the combination of LPS and TNFα although TNFα enhanced LPS-induced TF activity almost two fold. In contrast, when blood was incubated with LPS and PMA in whole blood, histamine gave a significant rise in TF activity at 0.01 µM and 0.33 µM histamine. The effect of histamine was less at 0.1 µM or higher concentrations giving a biphasic profile. Contrary to the effect of histamine on LPS plus PMA induced TF activity, histamine caused a significant reduction in TNFα albeit less than in the absence of PMA. Intake of aspirin caused a significant rise in LPS-induced TF activity that was almost abolished by histamine at 0.033 µM. CONCLUSION: Our study shows that histamine has an anti-inflammatory effect on LPS and LPS/TNFα stimulated monocytes of whole blood. In contrast when blood cells are activated by a combination of LPS and PMA whereby PKC is activated, histamine has a procoagulant/pro-inflammatory effect through enhancement of TF activity expression.

Antioxidant and anti-inflammatory activities of barettin.

In this paper, we present novel bioactivity for barettin isolated from the marine sponge Geodia barretti. We found that barettin showed strong antioxidant activity in biochemical assays as well as in a lipid peroxidation cell assay. A de-brominated synthetic analogue of barettin did not show the same activity in the antioxidant cell assay, indicating that bromine is important for cellular activity. Barettin was also able to inhibit the secretion of the inflammatory cytokines IL-1ß and TNFα from LPS-stimulated THP-1 cells. This combination of anti-inflammatory and antioxidant activities could indicate that barettin has an atheroprotective effect and may therefore be an interesting product to prevent development of atherosclerosis.

Circulating monocytes mirror the imbalance in TF and TFPI expression in carotid atherosclerotic plaques with lipid-rich and calcified morphology.

BACKGROUND: Thrombogenicity of atherosclerotic plaque largely depends on plaque morphology and their content of tissue factor (TF) and tissue factor pathway inhibitor (TFPI). The relationship between morphological composition of plaque (lipid-rich or calcified) and expression of TF and TFPI in circulating blood monocytes and within the plaques is not characterized. OBJECTIVE: To investigate whether lipid-rich (echolucent) or calcified (echogenic) morphology of carotid atherosclerotic plaques is associated with differences in TF and TFPI expression in circulating blood monocytes and within carotid atherosclerotic plaques. METHODS: We studied levels of monocyte TF and TFPI mRNA and protein expression and association with traditional risk factors for atherosclerosis in asymptomatic subjects with echolucent (n=20) or echogenic (n=20) carotid plaques, or controls without carotid atherosclerosis (n=20) determined by ultrasonography. Sections of calcified or lipid-rich carotid plaques obtained from symptomatic patients were assessed for TF and TFPI antigen expression. RESULTS: TF and TFPI surface presentation, surface TF/TFPI ratio, and TF activity were higher in monocytes obtained from subjects with echolucent than with echogenic plaques or controls without carotid atherosclerosis. Multiple regression analyses revealed inverse association between serum apoA1 and monocyte surface TF antigen expression (p=0.007), and positive association between serum apoB and monocyte surface TFPI expression (p=0.028). Sections from lipid-rich carotid plaques contained 2.5-fold more TF and 1.5-fold more TFPI antigens relative to calcified lesions, also yielding a higher TF/TFPI ratio. CONCLUSIONS: Our findings indicate that circulating monocytes of asymptomatic individuals with echolucent lipid-rich carotid atherosclerosis express an imbalance between TF and TFPI expression cohering with changes found within advanced carotid atherosclerotic plaques obtained from symptomatic patients.

Low thrombogenicity of calcified atherosclerotic plaques is associated with bone morphogenetic protein-2-dependent inhibition of tissue factor expression.

Morphology of atherosclerotic plaque is a major determinant of plaque thrombogenicity. Calcified atherosclerotic lesions are less prone to thrombosis and contain less tissue factor (TF) than lipid-rich plaques. Although bone morphogenetic protein (BMP)-2 is a known mediator of vascular calcification, the role of BMP-2 in the regulation of plaque thrombogenicity has not been established. We hypothesized that the expression of BMP-2 within highly calcified atherosclerotic plaques inhibits TF expression and reduces thrombogenicity of calcified lesions. In the present study, we measured levels of TF and BMP-2 in human calcified and lipid-rich carotid plaques and studied the effects of BMP-2 on TF expression in human monocytes in vitro. Quantitative immunohistochemical analysis of endarterectomy specimens for TF and BMP-2 revealed that calcified plaques contained nearly three-times less TF antigen than lipid-rich ones. In contrast, calcified plaques expressed two-times more BMP-2 antigen than lipid-rich lesions. BMP-2 markedly decreased protein expression and surface redistribution of TF in activated human monocytes in vitro. BMP-2-mediated inhibition of TF expression in monocytes/macrophages could contribute to reduced thrombogenicity of calcified atherosclerotic plaques.

Shear stress regulates inflammatory and thrombogenic gene transcripts in cultured human endothelial progenitor cells.

Shear stress has an established effect on mature endothelial cells, but less is known about how shear stress regulates endothelial progenitor cells (EPCs). In vitro expanded EPCs isolated from adult human blood represent a novel tool in regenerative vessel therapy. However, in vitro culturing may generate cells with unfavourable properties. The aim of the present study was therefore to assess whether shear stress may influence the inflammatory and thrombotic phenotype of in vitro expanded EPCs. In late outgrowth EPCs, 6 hours of shear stress (6.0 dynes/cm2) significantly reduced the mRNA levels of IL-8, COX2, and tissue factor (TF) compared to static controls. This was associated with a reduced TF activity. In contrast, mRNA expression of NOS3 was significantly increased following 6 and 24 hours of shear stress. In accordance with this, NOS3 protein expression was increased following 24 hours of shear stress. Overall stimulation with the proinflammatory mediator, TNFalpha, for the final 2 hours increased the mRNA expression of IL-6, IL-8, MCP-1, ICAM1, and TF. However exposure to 6 hours of shear stress significantly suppressed the inductory potential of TNFalpha to increase the mRNA levels of IL-6, IL-8, COX2, and TF. Additionally, TNFalpha increased TF activity approximately 10 times, an effect that was also significantly reduced by exposure to 6 and 24 hours of shear stress. The effect of shear on the gene levels of TF and NOS3 were not blocked by the NOS inhibitor L-NAME. These observations suggest that EPCs are capable of functionally responding to shear stress.

Soluble guanylate cyclase agonists inhibit expression and procoagulant activity of tissue factor.

OBJECTIVE: Tissue factor (TF), a major initiator of blood coagulation, contributes to inflammation, atherosclerosis, angiogenesis, and vascular remodeling. Pharmacological agonists of soluble guanylate cyclase (sGC) attenuate systemic and pulmonary hypertension, vascular remodeling, and platelet aggregation. However, the influence of these novel pharmacophores on TF is unknown. METHODS AND RESULTS: We evaluated effects of BAY 41-2272 and BAY 58-2667 on expression and activity of TF in human monocytes and umbilical vein endothelial cells (HUVECs). Both compounds reduced expression of active TF protein in monocytes stimulated with lipopolysaccharide, as demonstrated by immunoblotting and a TF procoagulant activity assay. In-cell Western assay revealed that this effect was associated with a marked reduction of total and surface TF presentation. Furthermore, BAY 41-2272 and BAY 58-2667 decreased TF protein expression and the TF-dependent procoagulant activity in HUVECs stimulated with TNF-alpha. The sGC agonists also suppressed transcriptional activity of NF-kappaB. A siRNA-mediated knockdown of the alpha1-subunit of sGC in monocytes and HUVECs confirmed that the inhibitory effect of BAY 41-2272 and BAY 58-2667 on TF expression is mediated through the sGC-dependent mechanisms. CONCLUSIONS: Inhibition of TF expression and activity by sGC agonists might provide therapeutic benefits in cardiovascular diseases associated with enhanced procoagulant and inflammatory response.

Seafood diets: hypolipidemic and antiatherogenic effects of taurine and n-3 fatty acids.

BACKGROUND: Health aspects of seafood have primarily been linked to n-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Although animal studies have suggested beneficial contributions from taurine, highly abundant in seafood, its effect in humans is obscure. This study evaluates the combined effects of n-3 PUFA and taurine. METHODS: Healthy volunteers (n=80) were recruited to a 7-week double-blind and parallel intervention trial. One group (n=39) received fish pâté (36g/day) enriched in n-3 (1.1gEPA+DHA/day) and the second (n=41) an identical pâté enriched both in n-3 and taurine (425mg/day). RESULTS: Total cholesterol (TC) (-5%, P<0.001), low-density lipoprotein (LDL)-cholesterol (-8%, P<0.001) and Apo B (-4%, P<0.001) decreased more in the n-3+taurine compared to the n-3 group. A significant within-group enhancement of high-density lipoprotein (HDL)-cholesterol was demonstrated in the n-3+taurine group (6%, P<0.0001). Reductions in triacylglycerol (TG) (-16%, P<0.05 in n-3; -14%, P<0.05 in n-3+taurine), thromboxane B(2) (TxB(2)) (-21%, P<0.001 in n-3; -15%, P<0.05 in n-3+taurine), tumor necrosis factor (TNFalpha) (-24%, P<0.001 in n-3; -12%, P<0.05 in n-3+taurine) and monocyte chemotactic protein (MCP-1) (-12%, P<0.05 in n-3; -6%, P<0.0001 in n-3+taurine) were evident in both groups. Reductions in interleukin (IL)-6 (-16%, P<0.05) and LTB(4) (-18%, P<0.05) were only significant in the n-3 group. CONCLUSIONS: The effects, particularly on blood lipids, of combining n-3 PUFA's and taurine proved superior to those of n-3 alone.

Enhanced incorporation of n-3 fatty acids from fish compared with fish oils.

This work was undertaken to study the impact of the source of n-3 FA on their incorporation in serum, on blood lipid composition, and on cellular activation. A clinical trial comprising 71 volunteers, divided into five groups, was performed. Three groups were given 400 g smoked salmon (n = 14), cooked salmon (n = 15), or cooked cod (n = 13) per week for 8 wk. A fourth group was given 15 mL/d of cod liver oil (CLO) (n = 15), and a fifth group served as control (n = 14) without supplementation. The serum content of EPA and DHA before and after intervention revealed a higher rise in EPA and DHA in the cooked salmon group (129% rise in EPA and 45% rise in DHA) as compared with CLO (106 and 25%, respectively) despite an intake of EPA and DHA in the CLO group of 3.0 g/d compared with 1.2 g/d in the cooked salmon group. No significant changes were observed in blood lipids, fibrinogen, fibrinolysis, or lipopolysaccharide (LPS)-induced tissue factor (TF) activity, tumor necrosis factor-alpha (TNFalpha), interleukin-8 (IL-8), leukotriene B4 (LTB4), and thromboxane B2 (TxB2) in whole blood. EPA and DHA were negatively correlated with LPS-induced TNFalpha, IL-8, LTB4, TxB2, and TF in whole blood. In conclusion, fish consumption is more effective in increasing serum EPA and DHA than supplementing the diet with fish oil. Since the n-3 FA are predominantly in TAG in fish as well as CLO, it is suggested that the larger uptake from fish than CLO is due to differences in physiochemical structure of the lipids.

The in vivo effect of melatonin on cellular activation processes in human blood during strenuous physical exercise.

Melatonin has been reported to have anti- as well as pro-inflammatory properties. Because physical stress is associated with the activation of blood cells, the present study examines melatonin's role in exercise-induced cell activation processes. Eight healthy volunteers (aged 20-62 yr, mean = 31), exercised on an 'Ergometric' bike for 30 min at 80% of their calculated maximum pulse rate. Blood samples were taken just before melatonin administration, directly after exercise, and 2 hr after exercise completion. Cytokine and eicosanoid parameters were measured in plasma from blood stimulated with lipopolysaccharide (LPS) for 2 hr whereas tissue factor (TF) activity was measured in isolated monocytes. Melatonin significantly decreased LPS-induced TF activity by 48% (P < 0.01) directly after exercise, and a 44% reduction was seen 2 hr later (P < 0.02). Furthermore, melatonin significantly reduced the lymphocyte count rise produced directly after exercising by more than 30% (P < 0.01). A trend was also seen for melatonin suppressing the increase of WBC by around 10% and to strengthen the platelet increase by about 8% after physical stress. Melatonin also significantly lowered RBC and hemoglobin counts by 5 and 3-4% during exercise (P < 0.005 and <0.02 respectively). Two hours after exercise, melatonin tended to lower leukotriene B4 levels by 30%. Interleukin-8 and tumor necrosis factor-alpha levels tended to be lower in individuals who had taken melatonin following hard physical activity and a larger sample size may show significance. Thromboxane B2 production seemed unaffected by melatonin during exercise. In conclusion, in vivo intake of melatonin appears to suppress LPS-induced activation of monocytes in whole blood reactions associated with physical exercise and facilitates the down-regulation of inflammatory mediators.

Generation of tissue factor-rich microparticles in an ex vivo whole blood model.

The aim of this study was to investigate the role of blood cells in the expression of tissue factor (TF) and P-selectin in platelets and microparticles from blood stimulated with lipopolysaccharide (LPS), with or without the further addition of phorbol myristyl acetate (PMA). TF activity was found to be associated with platelets after 2 h incubation of whole blood with LPS or LPS + PMA, while no TF activity was detected in microparticles from blood subjected to such stimulation. In blood stimulated for 6 and 24 h, addition of PMA to the samples led to a substantial increase in TF activity associated with microparticles, compared with stimulation with LPS alone. Addition of PMA to blood samples also led to a three-fold increase in the amount of P-selectin found in the isolated microparticle fraction, and a 50% reduction in P-selectin measured in platelets, compared with LPS alone used for stimulation. In a different experiment, TF-rich microparticles were shown to be absorbed very efficiently by neutrophils in a calcium-independent reaction. Our results imply that LPS stimulation of whole blood is associated with a direct transfer of TF from monocytes to platelets in the absence of free TF-rich microparticles, which probably is accounted for by the fusion of TF-rich microparticles with activated platelets exposing P-selectin. Further addition of PMA to samples generates both free TF-rich microparticles as well as enhanced transfer of TF from monocytes to platelets.

Intracellular and surface distribution of monocyte tissue factor: application to intersubject variability.

OBJECTIVE: The high and low responder phenomenon describes individual differences in lipopolysaccharide (LPS)-induced monocyte tissue factor (TF) activity. We characterized patterns of intracellular accumulation, externalization, and shedding of TF in response to LPS in mononuclear cells (MNCs) from high responders (HRs) and low responders (LRs). METHODS AND RESULTS: After 2 hours of LPS stimulation of whole blood, flow cytometry analyses revealed a larger population of TF-positive monocytes in HRs (32.0+/-3.5%) versus LRs (11.2+/-1.2%; P< or =0.05), along with a stronger mean fluorescence intensity of TF signal in HRs (7.1+/-0.5 arbitrary units [AU]) compared with LRs (5.4+/-0.4 AU; P< or =0.05). The LPS-treated blood of the HR group contained 2-fold more TF-positive microparticles than LRs. In-cell Western assay demonstrated higher intracellular accumulation of TF in mononuclear cells (MNCs) from LRs because LPS induced a 3.7-fold increase of total TF levels in LRs versus a 1.5-fold increase in HRs. In contrast, in response to LPS stimulation, MNCs from HRs exhibited a 4-fold induction of surface TF, whereas MNCs from LRs only had a minor increase in surface TF levels. CONCLUSIONS: The higher availability of surface TF antigen on MNCs from HRs and TF-containing microparticles might make these individuals more susceptible to hypercoagulation.

Tissue factor: (patho)physiology and cellular biology.

The transmembrane glycoprotein tissue factor (TF) is the initiator of the coagulation cascade in vivo. When TF is exposed to blood, it forms a high-affinity complex with the coagulation factors factor VII/activated factor VIIa (FVII/VIIa), activating factor IX and factor X, and ultimately leading to the formation of an insoluble fibrin clot. TF plays an essential role in hemostasis by restraining hemorrhage after vessel wall injury. An overview of biological and physiological aspects of TF, covering aspects consequential for thrombosis and hemostasis such as TF cell biology and biochemistry, blood-borne (circulating) TF, TF associated with microparticles, TF encryption-decryption, and regulation of TF activity and expression is presented. However, the emerging role of TF in the pathogenesis of diseases such as sepsis, atherosclerosis, certain cancers and diseases characterized by pathological fibrin deposition such as disseminated intravascular coagulation and thrombosis, has directed attention to the development of novel inhibitors of tissue factor for use as antithrombotic drugs. The main advantage of inhibitors of the TF*FVIIa pathway is that such inhibitors have the potential of inhibiting the coagulation cascade at its earliest stage. Thus, such therapeutics exert minimal disturbance of systemic hemostasis since they act locally at the site of vascular injury.

The effect of TNF-alpha, PMA, and LPS on plasma and cell-associated IL-8 in human leukocytes.

INTRODUCTION/AIM: This study was performed to examine the proficiency of mononuclear cells (MNC) and polymorphonuclear cells (PMN) in a whole blood model to expressing interleukin-8 (IL-8) in response to various stimuli. METHODS: Isolated cells that had been recombined with heparinized plasma were incubated with lipopolysaccharide (LPS), phorbol myristate acetate (PMA) and tumor necrosis factor (TNF)-alpha. RESULTS: IL-8 release by MNC was most potently induced by LPS, reaching significant levels after 2-h incubation in the presence of 0.2 ng/ml LPS. In contrast, 5.0 ng/ml LPS was required for PMN to release significant amounts of the cytokine (P<0.001). When PMN and MNC were coincubated (MNC/PMN), LPS-induced IL-8 release was reduced compared to the release from MNC alone, regardless of the concentration of LPS used. IL-8 release by PMN was much more strongly induced by TNF-alpha, increasing by 1050% in the presence of 10 ng/ml TNF-alpha (P<0.005), whereas MNC or MNC/PMN subjected to this stimulus alone did not significantly enhance their IL-8 release. PMA had no effect on IL-8 release from either cell type. Since a high portion of IL-8 in blood is associated with cells, the IL-8 levels in isolated and lysed cell suspensions were also quantified. Thus, a considerably higher level of IL-8 was found in freshly isolated PMN (0.58+/-0.09 ng/ml) than in MNC (0.010+/-0.007 ng/ml). PMN remained the main source for cell-associated IL-8 after 2-h incubation in the absence of any added stimuli, harbouring a relatively high level of the cytokine (3.37+/-1.38 ng/ml), which was significantly enhanced in the presence of TNF-alpha (8.99+/-1.46 ng/ml, P<0.001). CONCLUSION: This study shows that LPS is an effective inducer of IL-8 in MNC, whereas TNF-alpha is a potent agonist for IL-8 release from PMN. The main portion of cell-associated IL-8 is present in PMN when the cells are stimulated in their normal environment of plasma.

Effects of enalapril on disseminated intravascular thrombin formation during systemic inflammation.

BACKGROUND: Tissue factor (TF), the main trigger of coagulation is important in the propagation of cardiovascular diseases. Based on an in vitro study, we hypothesised that enalapril may blunt the endotoxin-induced, TF-triggered coagulation in humans. METHODS: In a randomised, controlled trial, 30 healthy male volunteers received 2 ng/kg of lipopolysaccharide (LPS) after pre-treatment with placebo or enalapril for 5 days or with enalapril 2 h before LPS infusion. RESULTS: Infusion of LPS increased interleukin-6 levels 400 fold, and induced a 10-fold increase in prothrombin fragment, a fourfold increase in D-dimer, and a fivefold increase in plasmin-antiplasmin complexes. However, pre-treatment with enalapril did not blunt LPS-induced coagulation. CONCLUSIONS: Our trial provides evidence against a modulatory role of angiotensin converting enzyme in LPS-induced, TF-triggered coagulation.