Increased Tissue Factor Activity Research Articles

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.

Glycogen Synthase Kinase-3 Mediates Endothelial Cell Activation by Tumor Necrosis Factor-α

Endothelial cell transformation to a thrombogenic and inflammatory phenotype plays an important role in the pathogenesis of atherothrombosis, but the responsible signaling pathways remain to be elucidated. This study was designed to investigate the regulatory role of glycogen synthase kinase-3 (GSK-3) in tissue factor (TF) and vascular cell adhesion molecule (VCAM)-1 expression in tumor necrosis factor (TNF)-alpha-stimulated endothelial cells. In human endothelial cells, TNF-alpha as well as thrombin induced rapid and transient dephosphorylation and hence, activation of GSK-3. A GSK-3 inhibitor, LiCl, suppressed TNF-alpha- and thrombin-induced TF and VCAM-1 expression, whereas NaCl had no effect. A specific GSK-3 inhibitor, TDZD-8, mimicked the inhibitory effects of lithium. GSK-3 inhibition also significantly suppressed the TNF-alpha-induced increase in TF activity and VCAM-1 cell-surface expression. The luciferase reporter system demonstrated that regulation of TF and VCAM-1 expression by GSK-3 was mediated at the transcriptional level. The TNF-alpha-induced increase in nuclear factor (NF)-kappaB DNA-binding activity was significantly suppressed by TDZD-8. TDZD-8 completely prevented the TNF-alpha-induced inhibitor of NF-kappaB (IkappaB)-alpha degradation but had no effect on IkappaB-kinase-beta phosphorylation. GSK-3 regulates TNF-alpha-induced IkappaB-alpha degradation and NF-kappaB activation independent of IkappaB-kinase-beta and subsequent induction of TF and VCAM-1 expression in human endothelial cells. This study provides the experimental basis for a novel strategy of using GSK-3 inhibition to treat atherothrombotic vascular disease.

Sirolimus increases tissue factor expression but not activity in cultured human vascular smooth muscle cells

BackgroundSirolimus-eluting stents (CYPHER stents) demonstrated remarkable efficacy in reducing restenosis rates in patients with coronary artery disease. There is a concern of sub-acute and late stent thrombosis. Tissue factor (TF) is critical in thrombosis. This study investigated the effect of sirolimus on TF expression and activity in cultured human vascular smooth muscle cells (SMCs).MethodsSMCs were cultured from human saphenous veins and aortas. Quiescent cells were stimulated with sirolimus (0.1 – 20 ng/ml) over 24 hours. Cellular TF expression and activity released into culture medium were measured. The effect of sirolimus on activation of mammalian target of rapamycin (mTOR) was measured by phosphorylation of the substrate p70s6k at T389, and activation of RhoA was measured by pull-down assay.ResultsSirolimus increased TF protein level in cultured human SMCs in a concentration and time-dependent manner (about 2-fold, p < 0.01) reaching maximal effect at 5 ng/ml. The stimulation of TF expression by sirolimus was associated with inhibition of basal activity of mTOR. No effects of sirolimus on RhoA or p38mapk activation that are positive regulators of TF in vascular wall cells were observed. The stimulation of TF expression by sirolimus (20 ng/ml) was prevented by the HMG-CoA reductase inhibitor fluvastatin (1 μmol/L). However, no increase in TF activity released from SMC into culture medium was observed after sirolimus treatment.ConclusionAlthough sirolimus stimulates TF protein expression in human SMC associated with inhibition of mTOR, it does not enhance TF activity released from the cells, suggesting a relatively safe profile of CYPHER stents. The inhibition of TF expression by fluvastatin favors clinical use of statins in patients undergoing coronary stenting.

Up-regulation of tissue factor activity on human proximal tubular epithelial cells in response to Shiga toxin

The pathophysiology of hemolytic uremic syndrome (HUS) is incompletely established. Based on clinical studies demonstrating the presence of prothrombotic plasma markers in patients with HUS, we hypothesized that Shiga toxin might cause activation of the coagulation pathway by augmenting tissue factor, the major initiator of coagulation. Human proximal tubular epithelial cells (PTECs) [human kidney-2 (HK-2 cells)] were exposed to Shiga toxin-1, and expression of tissue factor, cell detachment, protein synthesis, caspase-3 activity, and Shiga toxin-1 binding were examined. Results. HK-2 cells expressed constitutive surface tissue factor activity and increased their tissue factor expression upon exposure to Shiga toxin-1. Shiga toxin-1 bound to HK-2 cells and inhibited protein synthesis. The up-regulation of tissue factor was dose- and time-dependent and strongly correlated with cell detachment and increase in caspase-3 activity caused by Shiga toxin-1 exposure. A general caspase inhibitor simultaneously inhibited HK-2 cell detachment and tissue factor up-regulation while mutant Shiga toxin-1 neither caused cell detachment, protein synthesis inhibition, nor increase in tissue factor activity. Tissue factor activity elicited by Shiga toxin-1 was abrogated by a monoclonal antitissue factor antibody. Calphostin C, a protein kinase C (PKC) inhibitor, partially blocked tissue factor up-regulation, indicating possible involvement of PKC-dependent mechanism. These data, taken together, suggest a strong link between Shiga toxin-induced up-regulation of tissue factor activity, cytotoxicity, and apoptosis in HK-2 cells. The proximal tubule is a target of Shiga toxin in HUS, and it seems plausible that injured proximal tubular cells trigger the activation of the coagulation system, the formation of intrarenal platelet-fibrin thrombi, and the development of acute renal failure in HUS.

Detached endothelial cells and microparticles as sources of tissue factor activity

Introduction Cytokine activation of endothelial cell monolayers is associated with cell detachment, microparticle shedding from plasma membranes, and phosphatidylserine appearance in the plasma membrane outer leaflets. While tissue factor expression on activated endothelial cells and microparticles is well documented, the contribution of detached endothelial cells to tissue factor activity is less clear. We studied tissue factor expression and the role of tissue factor pathway inhibitor on adherent and detached endothelial cells and on microparticles following endothelial cell activation with TNF-α. Materials and methods Detached endothelial cells and microparticles were obtained from cultures of human umbilical vein endothelial cells by differential centrifugation of cell culture supernatant. For microparticle capture, an antibody directed against CD146 was used. Functional tissue factor activity was measured by chromogenic assay and tissue factor antigen by ELISA. Endothelial cell and microparticle morphology was examined by light and transmission electron microscopy. Results After cell activation for 22 h, functional tissue factor activity was distributed as follows: 60%, adherent endothelial cells; 35%, detached cells; and 5%, microparticles. Tissue factor protein followed a similar distribution. Cell detachment was 47%. Electron microscopy demonstrated shedding of microparticles with a diameter of 0.1–0.6 μm. Cy3–annexin V revealed increased phosphatidylserine on activated adherent endothelial cells and microparticles. Pre-incubation of adherent and detached endothelial cells and microparticles with anti-tissue factor antibody blocked factor Xa production. Pre-incubation with anti-tissue factor pathway inhibitor antibody increased tissue factor activity of adherent endothelial cells 2.8-fold, detached cells 1.4-fold, and microparticles 45-fold. Conclusions Detached endothelial cells as well as microparticles from activated endothelial cell monolayers express tissue factor activity, and this activity on microparticles is markedly inhibited by microparticle-associated tissue factor pathway inhibitor.

The thrombogenic effect of an inflammatory cytokine on trophoblasts from women with preeclampsia

This study was undertaken to investigate whether the increased thrombogenic potential of cytotrophoblastic cells of women with preeclampsia can be accounted for by increased rates of apoptosis. Cytotrophoblasts were isolated from the placenta of (a) nulliparous women without hypertensive disease who were delivered at term and (b) nulliparous women with preeclampsia. The cytotrophoblasts were identified by morphology, and cytokeratin and gonadotropin-releasing hormone positivity. The inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) was added to cytotrophoblasts in vitro and incubated for 24 hours. Tissue factor antigen, activity, and amount of apoptosis were evaluated both before and after TNF-alpha stimulation. TNF-alpha simulation significantly increased tissue factor activity both in the cytotrophoblasts of women with (0.08 +/- 0.04 pmol/min/10 6 cells to 0.53 +/- 0.19 pmol/min/10 6 cells) and without (0.07 +/- 0.04 pmol/min/10 6 cells to 0.30 +/- 0.16 pmol/min/10 6 cells) preeclampsia. TNF-alpha stimulation of the cytotrophoblasts also significantly increased tissue factor antigen in the cytotrophoblasts of both groups of women (3.6 +/- 0.9 fmol/10 6 cells to 34.0 +/- 7.5 fmol/10 6 cells, and 7.5 +/- 1.4 fmol/10 6 cells to 25.4 +/- 2.2 fmol/10 6 cells, respectively). For both tissue factor antigen and activity, the magnitude of increase after stimulation was significantly greater in the preeclamptic cytotrophoblasts. In contrast, both normal and preeclamptic cytotrophoblasts showed similar increases in their apoptotic indices (approximately 2-fold) after induction by TNF-alpha. The greater response of tissue factor activity and antigen to TNF-alpha by preeclamptic cytotrophoblasts cannot be accounted for by the increase in apoptosis. These data suggest that preeclamptic cytotrophoblasts are inherently more thrombogenic and more sensitive to TNF-alpha stimulation.

The Principal Eosinophil Peroxidase Oxidant, HOSCN, Potently Induces Endothelial Cell Tissue Factor Activity through Transcriptional Activation Dependent upon NF-kB and ERK1/2: Potential Mechanism for a Prothrombotic and Proinflammatory Endothelial Phenotype in the Hypereosinophilic Syndrome.

We have shown that SCN- is the principal substrate for EPO in vivo and its product, HOSCN, a weak sulfhydryl-reactive oxidant, is a uniquely potent (i.e. 10–1000-fold more than any other) phagocyte oxidant inducer of tissue factor (TF) activity in human umbilical vein endothelial cells (HUVEC). We here characterize the intracellular signaling pathways and transcription factors activated by HUVEC in response to this unusual oxidant. We exposed monolayers to increasing concentrations of various phagocyte-derived oxidants or 10 ng/ml LPS in M199 medium with 10% FCS and assayed TF in scraped, freeze-thawed extracts by one-stage clotting assay. We find that 50–150 μM HOSCN induces TF activity up to 100-fold at 4–6 hours, levels similar to those stimulated by LPS. In contrast, similar concentrations of HOBr, H2O2, and HOCl induced at most a < 2-fold increase in TF activity. Because the TF gene has upstream regulatory sequences that bind transcription factors AP-1, Egr-1 and NF-kB, we probed nuclear extracts of HOSCN- and LPS-treated HUVEC by EMSA using oligonucleotide probes specific for their respective factors. HOSCN induces potent (similar to 10 μg/ml LPS/ml) upregulation of NF-kB p50/p65 and the TF-specific c-rel/p65 heterodimer by 1–2 hours as confirmed by supershift assay. HOSCN induced modest activation of Egr-1 at 1 hour and strong activation of the c-Jun but not c-Fos AP-1 binding factors. Investigating intracellular kinase pathways upstream from these transcription factors, we probed Western blots of HOSCN-treated HUVEC extracts for the phosphorylated forms of ERK1/2 and SAPK/JNK and for I-kB. HOSCN stimulates a transient increase in pERK1/2 at 10–20 min followed by a pronounced secondary increase starting at 1h and peaking at 2–4 h as well as increasing pSAPK/JNK at 1-4h and promoting degradation of I-kB. The functional significance of ERK1/2 and NF-kB was confirmed using specific inhibitors of these respective pathways, U0126 and andrographolide, either of which nearly completely blocked HOSCN-induced TF expression. The critical role of NF-kB in this phenomenon was shown when wortmannin, which blocks the PI3K/Akt pathway that constitutively inhibits NF-kB, increased TF activity 10–30 fold but simultaneous addition of andrographolide blocked this increase. To extend our analysis of HOSCN on transcriptional activation, we screened 54 separate transcription factors using a Panomics TranSignal™Protein/DNA array and found that HOSCN induces >3-fold activation of Stat4, CDP, GRE, CBF, and Ets-1/PEA3, a pattern similar to but readily distinguishable from that of LPS. We conclude HOSCN strongly induces TF activity in endothelial cells through transcriptional activation dependent upon ERK 1/2 and NF-kB. Given the widespread genomic distribution of binding sites for these transcription factors and the types of genes they regulate, we hypothesize HOSCN generated by adherent and infiltrating eosinophils may provoke the development of a prothrombotic and proinflammatory endothelial/endocardial phenotype that promotes the pronounced thrombotic diathesis characteristic of the Hypereosinophilic Syndrome.

The long pentraxin PTX3 up-regulates tissue factor in activated monocytes: another link between inflammation and clotting activation.

Pentraxin-3 (PTX3), an acute-phase protein that belongs to the family of the PTXs, is found elevated in septic shock and increased in patients with acute myocardial infarction. As tissue factor (TF) plays a key role in thrombosis and inflammation associated with atherosclerosis and as we have recently reported that PTX3 increases TF synthesis in endothelial cells, we tested whether PTX3 could modulate TF expression in monocytes. Monocytes from peripheral blood of healthy donors were incubated with highly purified PTX3 with or without lipopolysaccharide (LPS). Cells were then disrupted, and procoagulant activity was assessed by a one-stage clotting time. PTX3 enhanced TF activity and antigen from LPS-stimulated monocytes in a dose-dependent way. The effect was specific, as other PTXs, such as C-reactive protein and serum amyloid P component, were ineffective. Moreover, the increase in activity was specific for LPS, as in the presence of other TF-inducing agents such as interleukin-1beta and tumor necrosis factor alpha, PTX3 was not effective. The increase in TF activity requires mRNA synthesis, as assessed by polymerase chain reaction. The mechanism by which PTX3 modulates TF synthesis resides in an enhanced IkappaB, alpha phosphorylation and degradation and increased migration of the transacting factor c-Rel/p65 into the nucleus, as determined by Western blot and electro-mobility shift assay. These results show that PTX3 is an enhancer of the expression of TF by mononuclear cells. In the area of vascular injury, during the inflammatory response, cell-mediated fibrin deposition takes place. PTX3 increases TF expression, thus potentially playing a role in thrombogenesis and wound healing.

Oxidized lipoprotein regulation of tissue factor in smooth muscle cells

Cell surface tissue factor (TF), which can initiate local coagulation, is upregulated in cells within atherosclerotic plaques, including macrophages, endothelial cells (EC) and smooth muscle cells (SMC). TF binds coagulation factors VIIa and Xa to make a catalytically active complex that produces thrombin and ultimately fibrin, leading to inflammation and coagulation. Using rat SMC, we have shown that lipids of low-density lipoprotein (LDL) and oxidized LDL (oxLDL) differentially regulate TF gene expression and TF activity. Unknown lipids of LDL induced the gene, but did not enhance activity. In contrast, oxLDL or its extracted lipids induced both the TF gene and TF activity. Specific lipids of oxLDL, e.g., lipid hydroperoxides, and H 2O 2 increased TF activity without enhancing TF gene or protein. The effects of lipid hydroperoxides were antioxidant-inhibitable. Lysophosphatidic acid (lysoPA) and lysophosphatidylcholine (lysoPC), important lipid mediators of many cellular processes and formed during LDL oxidation, increased TF mRNA, protein and activity. LysoPA induced the TF gene via a Gi-protein-coupled signaling process. Thus, different lipoprotein-borne lipids in atherosclerotic lesions enhance TF gene expression and TF activity through distinct mechanisms. These effects may contribute to lesion development and subsequent complications.

Interactions of Aspergillus fumigatus with endothelial cells: internalization, injury, and stimulation of tissue factor activity

AbstractInvasive aspergillosis causes significant mortality among patients with hematologic malignancies. This infection is characterized by vascular invasion and thrombosis. To study the pathogenesis of invasive aspergillosis, we investigated the interactions of Aspergillus fumigatus conidia and hyphae with endothelial cells in vitro. We found that both forms of the organism induced endothelial cell microfilament rearrangement and subsequent endocytosis. Conidia were endocytosed 2-fold more avidly than hyphae, and endocytosis was independent of fungal viability. Endocytosed conidia and hyphae caused progressive endothelial cell injury after 4 hours of infection. Live conidia induced more endothelial cell injury than did live hyphae. However, endothelial cell injury caused by conidia was dependent on fungal viability, whereas injury caused by hyphae was not, indicating that conidia and hyphae injure endothelial cells by different mechanisms. Neither live nor killed conidia increased tissue factor activity of endothelial cells. In contrast, both live and killed hyphae stimulated significant endothelial cell tissue factor activity, as well as the expression of tissue factor antigen on the endothelial cell surface. These results suggest that angioinvasion and thrombosis caused by A fumigatus hyphae in vivo may be due in part to endothelial cell invasion, induction of injury, and stimulation of tissue factor activity.

Mechanisms of autoantibody-induced monocyte tissue factor expression

The expression of tissue factor (TF) activity to flowing blood is the trigger for physiological coagulation as well as many types of thrombosis. A growing body of evidence suggests that increased tissue factor activity is a significant contributor towards the hypercoagulability associated with the antiphospholipid syndrome (APS). The increase in tissue factor activity appears to be due to increased transcription and translation of nascent tissue factor molecules but is not due to de-encryption of existing tissue factor molecules on cells. Autoantibodies and/or immune complexes circulating in APS patients appear to enhance the expression of tissue factor activity on monocytes and endothelial cells. Anti-β 2-glycoprotein I (β2GPI) autoantibodies have been specifically implicated in the antibody-mediated enhancement of tissue factor activity. The presence of antibodies against tissue factor pathway inhibitor (TFPI) in certain APS patients suggests that negative regulation of tissue factor activity might also be impaired in these patients. Given a mechanism involving increased tissue factor activity in APS-associated thrombosis, agents specifically targeting tissue factor activity may be a novel and efficacious therapy that is safer than current approaches to the management of APS.

Regulation of tissue factor expression in smooth muscle cells with nitric oxide

This study was undertaken to determine the effect of nitric oxide (NO) on tissue factor (TF) expression in vascular smooth muscle cells. Rat aortic smooth muscle cells (RASMCs) were exposed to NO delivered exogenously with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) or produced endogenously after infection with an adenoviral vector carrying human inducible NO synthase (AdiNOS). Functional TF activity was assessed with chromogenic TF assay. TF antigen was determined with immunohistochemistry. Northern blot analysis was used to determine steady- state TF messenger RNA (mRNA). Electrophoretic mobility gel shift assay was performed to determine the nuclear binding activity of nuclear factor kappa-B (NFkappaB). NFkappaB activity was inhibited by either prior transduction of RASMCs with mutant IkappaB or treatment with pyrrolidine dithiocarbamate. RASMCs exposed to SNAP or infected with AdiNOS exhibited increased functional TF activity and antigen. Regardless of the source of NO, a time-dependent and concentration-dependent increase in TF activity was observed. Steady-state TF mRNA levels were also increased by NO delivered via either method. NFkappaB nuclear binding activity was also increased by NO. Inhibition of NFkappaB activity by either pyrrolidine dithiocarbamate treatment or mutant IkappaB transduction abrogated NO-induced enhancement of TF mRNA and functional activity. In RASMC, NO exposure results in upregulation of TF functional activity, antigen, and mRNA. This effect appears to be mediated by an NFkappaB-dependent pathway.

P1708 The omega-3 fatty acid docosahexaenoate increases tissue factor activity despite in surface expression in activated human endothelial cells

P1708 The omega-3 fatty acid docosahexaenoate increases tissue factor activity despite in surface expression in activated human endothelial cells

Effect of hormone replacement therapy on tissue factor activity, C-reactive protein, and the tissue factor pathway inhibitor

W have previously observed that hormone replacement therapy (HRT) significantly increased tissue factor (TF) activity in healthy women1 and tended to increase prothrombin fragment 1 2 levels in 20 hypertensive and/or overweight postmenopausal women.2 However, we did not measure other indexes of coagulation activation or inhibition, nor did we measure C-reactive protein (CRP), which in experimental preparations stimulates the synthesis and release of the coagulation activator TF.3 TF pathway inhibitor (TFPI) may interact with TF to maintain homeostasis.4,5 Thus, this study examines (1) whether HRT increases TF activity, which may facilitate thrombin formation in hypertensive and/or overweight postmenopausal women, and (2) whether HRT-induced TF activity is associated with changes in CRP or TFPI that might provide mechanistic insight. • • • Thirty postmenopausal women (mean SD 59 6 years) participated in this study. Inclusion criteria have been previously reported.2 Eight, 8, and 14 women were overweight, hypertensive, and both, respectively. Seven of 22 were obese. Mean body mass index was 28.7 3.4. We treated hypertensive women with a low salt diet and/or diuretics (hydrochlorothiazide or furosemide) alone to avoid drug effects on hemostasis. This study was randomized, double-blind, and crossover in design. Study participants received micronized progesterone (MP) 100 mg or medroxyprogesterone acetate (MPA) 2.5 mg with conjugated equine estrogen (CEE) 0.625 mg daily for 2 months with the second treatment period initiated upon completion of the first treatment period. The study was approved by the Gil Hospital institute review board and all participants gave written, informed consent. Assays for hemostasis were performed as previously described.6,7Blood samples for laboratory assays were obtained from an antecubital vein after an overnight fast, before and at the end of treatment. Because of the circadian variation of fibrinolytic activity, blood collection was restricted to the hours of 8 to 9 A.M. We measured parameters of coagulation system activation. Prothrombin fragment 1 2 and thrombin-antithrombin complexes were measured by enzyme-linked immunosorbent assays (ELISA) (Behring Diagnostics Inc., San Jose, California). TF and TFPI antigen and activity were measured in duplicate by ELISA and actichrome assays (American Diagnostica, Greenwich, Connecticut). In all patients, serum was collected for the measurement of CRP levels. CRP levels were determined with an immunonephelometry system according to methods described by the manufacturer (Rate nephelometry; IMMAGE, Beckman Coulter, Brea, California). The measurement range was 0.1 to 98 mg/dl. All samples from the same patient (batch samples) were measured in blinded pairs with the same ELISA kit to minimize run-to-run variability. The interand intraassay coefficients of variation were 8%. Data are expressed as mean SEM or median (range 25% to 75%). After testing data for normality, we used Student’s paired t test or the Wilcoxon signed-rank test to compare values at baseline and after each therapy, as reported in Table 1. We assumed that the second baseline after the washout would not be different from the first baseline, because we saw no carryover effect of CEE and progestagen after 6 weeks in our previous studies.6,8 Thus, we decided on a treatment period of 2 months without washout. The effects of the 2 therapies on markers of hemostasis relative to baseline values were analyzed by 1-way repeated measures analysis of variance or Friedman’s repeated analysis of variance on ranks. After demonstration of significant differences among therapies by analysis of variance, post hoc comparisons between treatment pairs were made using the Student-Newman-Keuls multiple comparison procedures. Pearson’s correlation coefficient analysis was used to assess associations between measured parameters. In light of the multiple comparisons generated by the exploratory nature of this study, a p value 0.001 (rather than 0.05) was used to indicate statistical significance. There were no carryover effects from the initial treatment periods to the next treatment period. The effects of therapies on hemostasis are shown in Table 1. CEE MP and CEE MPA therapies tended to increase CRP levels from baseline values (p 0.107 by analysis of variance). Both therapies significantly decreased TF antigen but increased TF activity from From the Departments of Cardiology, Clinical Pathology, and Preventive Medicine (Biostatistics), Gachon Medical School, Incheon, Korea. This study was supported by grants (2000-1-20500-1) from the Basic Research Program, Korea Science and Engineering Foundation, Taejeon, Korea. Dr. Koh’s address: Vascular Medicine and Atherosclerosis Unit, Cardiology, Gil Heart Center, Gachon Medical School, 1198 Kuwol-dong, Namdong-gu, Incheon, Korea 405-760. E-mail: kwangk@ghil.com. Manuscript received July 15, 2002; revised manuscript received and accepted September 19, 2002.

Anisodamine inhibits endotoxin-induced tissue factor expression in human endothelial cells.

By study on the effect of anisodamine on lipopolysaccharide-induced expression of tissue factor (TF) in vascular endothelial cells (EC), the mechanism of anisodamine antithrombosis, as well as in the treatment of bacteraemic shock was investigated. Human umbilical vein endothelial cells (HUVECs) were cultured by trypsin digestion method. TF activity was measured in the lysates of HUVEC by using a single step clotting assay. Specific mRNA expression was detected by Northern blotting. In order to evaluate a possible contribution of the nuclear factor (NF)-kappa B pathway on the effects observed, electrophoretic mobility shift assays (EMSA) were performed using nuclear extracts from HUVECs and NF-kappa B-binding oligonucleotides. The results showed that treatment of HUVEC with LPS resulted in a significant increase in TF activity. Anisodamine dose-dependently inhibited LPS-induced upregulation of TF. These effects was also confirmed on the level of specific TF mRNA expression by Northern blotting. Furthermore, EMSA showed that anisodamine completely abolished LPS-induced NF-kappa B DNA binding activity in nuclear extracts from HUVECs treated with LPS together with anisodamine. The results suggest that anisodamine counteracts endothelial cell activation by inhibiting LPS-induced 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 effect on endothelial cells might be one underlying mechanism explaining its antithrombosis and 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.

Statin prevents tissue factor expression in human endothelial cells: role of Rho/Rho-kinase and Akt pathways.

Tissue factor plays a pivotal role in thrombus formation in acute coronary syndromes. However, the regulatory mechanisms underlying tissue factor expression are poorly understood. Statins are effective in patients with acute coronary syndromes. Hence, the aim of this study was to clarify in human endothelial cells the signaling pathways of thrombin-induced tissue factor expression and potential inhibitory effects of statins. In human aortic endothelial cells, simvastatin prevented tissue factor induction by thrombin (4 U/mL) in a concentration-dependent manner. The increase in tissue factor activity on the cell surface was also blocked by simvastatin. Simvastatin also prevented the upregulation of tissue factor expression and activity in human aortic smooth muscle cells. Mevalonate (100 micromol/L) reversed the inhibitory effect of simvastatin on tissue factor expression. Thrombin induced rapid activation of Rho A and p38 MAP kinase. The Rho-kinase inhibitor Y-27632 and the p38 MAP kinase inhibitor SB203580 prevented tissue factor induction. Akt was dephosphorylated by thrombin; the phosphoinositol 3-kinase inhibitor wortmannin enhanced its dephosphorylation as well as thrombin-induced tissue factor expression. Simvastatin prevented thrombin-induced Rho A activation but not p38 MAP kinase activation. Akt dephosphorylation by thrombin was blocked by both simvastatin and Y-27632. Endothelial tissue factor induction by thrombin is regulated by Rho/Rho-kinase, Akt, and p38 MAP kinase. Simvastatin prevents its induction through inhibition of Rho/Rho-kinase and activation of Akt. These findings provide new insights into the action of statins in acute coronary syndromes.

Effects of hormone replacement therapy on coagulation and fibrinolysis in postmenopausal women.

It has been speculated that hormone replacement therapy (HRT) containing relatively low dose of estrogen would be different from oral contraceptive pills in causing thromboembolism because activation of coagulation depends on the amount of estrogen. In contrast to this knowledge, activation of coagulation pathways has been detected in postmenopausal women treated with HRT in the observational and clinical studies. In this regard, recent studies have reported a 2 to approximately 4 fold risk of venous thromboembolism or pulmonary embolism in postmenopausal women receiving HRT than in non-users of estrogen. On the other hands, HRT has shown to enhance systemic fibrinolysis with decreased plasma plasminogen activator inhibitor-1 (PAI-1) levels. In addition, levels of D-dimer exhibited a significant inverse correlation with PAI-1 levels, suggesting enhanced fibrinolysis potential. However, small doses of estrogen/progestogen induce increases in fibrinolytic capacity via a marked reduction of PAI-1. In other words, HRT at conventional dosages may affect fibrinolytic activity to a greater extent than coagulation activity, whereas the converse trend holds at higher estrogen doses. The increase in fibrinolytic potential was independent of any effect on coagulation of CEE at conventional dosages. However, in contrast to healthy postmenopausal women, we recently reported that HRT did not significantly decrease PAI-1 antigen levels and rather, increased tissue factor activity and prothrombin fragment F(1+2) levels from baseline in hypertensive and/or overweight postmenopausal women. Activation of coagulation following HRT may not be balanced by activation of fibrinolysis in some postmenopausal women. Thrombogenic events are considered more likely in patients with certain heritable conditions, such as platelet antigen-2 (PIA-2) polymorphisms. Further, Factor V Leiden mutation increases the risk of primary and recurrent venous thromboembolic events by three to sixfold and the risk of myocardial infarction. Indeed, HRT may decrease or increase atherothrombosis risk depending on the presence of Factor V Leiden mutation. Thus, HRT should not be initiated in women with established coronary artery disease or the coexistence of other risk factors for hypercoagulability-malignancy, immobility, obesity, diabetes, advanced age, or inherited traits. However, HRT at conventional dosages improves fibrinolysis potential in healthy postmenopausal women.

Long pentraxin PTX3 upregulates tissue factor expression in human endothelial cells: a novel link between vascular inflammation and clotting activation.

Inflammation is a major contributing factor to atherosclerotic plaque development and ischemic heart disease. PTX3 is a long pentraxin that was recently found to be increased in patients with acute myocardial infarction. Because tissue factor (TF), the in vivo trigger of blood coagulation, plays a dominant role in thrombus formation after plaque rupture, we tested the possibility that PTX3 could modulate TF expression. Human umbilical vein endothelial cells, incubated with endotoxin (lipopolysaccharide) or the inflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha, expressed TF. The presence of PTX3 increased TF activity and antigen severalfold in a dose-dependent fashion. PTX3 exerted its effect at the transcription level, inasmuch as the increased levels of TF mRNA, mediated by the stimuli, were enhanced in its presence. The increase in mRNA determined by PTX3 originated from an enhanced nuclear binding activity of the transacting factor c-Rel/p65, which was mediated by the agonists and measured by electrophoretic mobility shift assay. The mechanism underlying the increased c-Rel/p65 activity resided in an enhanced degradation of the c-Rel/p65 inhibitory protein IkappaBalpha. In the area of vascular injury, during the inflammatory response, cell-mediated fibrin deposition takes place. Our results suggest that PTX3, by increasing TF expression, potentially plays a role in thrombogenesis and ischemic vascular disease.

Vascular effects of estrogen in type II diabetic postmenopausal women

OBJECTIVESWe assessed the effects of estrogen on vascular dilatory and other homeostatic functions potentially affected by nitric oxide (NO)-potentiating properties in type II diabetic postmenopausal women.BACKGROUNDThere is a higher cardiovascular risk in diabetic women than in nondiabetic women. This would suggest that women with diabetes do not have the cardioprotection associated with estrogen.METHODSWe administered placebo or conjugated equine estrogen, 0.625 mg/day for 8 weeks, to 20 type II diabetic postmenopausal women in a randomized, double-blinded, placebo-controlled, cross-over design.RESULTSCompared with placebo, estrogen tended to lower low-density lipoprotein (LDL) cholesterol levels by 15 ± 23% (p = 0.007) and increase high-density lipoprotein (HDL) cholesterol levels by 8 ± 16% (p = 0.034). Thus, the ratio of LDL to HDL cholesterol levels significantly decreased with estrogen, by 20 ± 24%, as compared with placebo (p = 0.001). Compared with placebo, estrogen tended to increase triglyceride levels by 16 ± 48% and lower glycosylated hemoglobin levels by 3 ± 13% (p = 0.295 and p = 0.199, respectively). However, estrogen did not significantly improve the percent flow-mediated dilatory response to hyperemia (17 ± 75% vs. placebo; p = 0.501). The statistical power to accept our observation was 81.5%. Compared with placebo, estrogen did not significantly change E-selectin, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, monocyte chemoattractant protein-1 or matrix metalloproteinase-9 levels. Compared with placebo, estrogen tended to decrease tissue factor antigen and increase tissue factor activity levels by 7 ± 46% and 5 ± 34%, respectively (p = 0.321 and p = 0.117, respectively) and lower plasminogen activator inhibitor-1 levels by 16 ± 31% (p = 0.043).CONCLUSIONSThe effects of estrogen on endothelial, vascular dilatory and other homeostatic functions were less apparent in type II diabetic postmenopausal women, despite the beneficial effects of estrogen on lipoprotein levels.