Thrombin-induced Tissue Factor Research Articles

PAR2 Activation on Human Kidney Tubular Epithelial Cells Induces Tissue Factor Synthesis, That Enhances Blood Clotting.

Coagulation abnormalities and increased risk of atherothrombosis are common in patients with chronic kidney diseases (CKD). Mechanisms that alter renal hemostasis and lead to thrombotic events are not fully understood. Here we show that activation of protease activated receptor-2 (PAR2) on human kidney tubular epithelial cells (HTECs), induces tissue factor (TF) synthesis and secretion that enhances blood clotting. PAR-activating coagulation-associated protease (thrombin), as well as specific PAR2 activators (matriptase, trypsin, or synthetic agonist 2f-LIGRLO-NH2 (2F), induced TF synthesis and secretion that were potently inhibited by PAR2 antagonist, I-191. Thrombin-induced TF was also inhibited by a PAR1 antagonist, Vorapaxar. Peptide activators of PAR1, PAR3, and PAR4 failed to induce TF synthesis. Differential centrifugation of the 2F-conditoned medium sedimented the secreted TF, together with the exosome marker ALG-2 interacting protein X (ALIX), indicating that secreted TF was associated with extracellular vesicles. 2F-treated HTEC conditioned medium significantly enhanced blood clotting, which was prevented by pre-incubating this medium with an antibody for TF. In summary, activation of PAR2 on HTEC stimulates synthesis and secretion of TF that induces blood clotting, and this is attenuated by PAR2 antagonism. Thrombin-induced TF synthesis is at least partly mediated by PAR1 transactivation of PAR2. These findings reveal how underlying hemostatic imbalances might increase thrombosis risk and subsequent chronic fibrin deposition in the kidneys of patients with CKD and suggest PAR2 antagonism as a potential therapeutic strategy for intervening in CKD progression.

Anti-thrombotic properties of a CD31-derived peptide coated on a surface

Event Abstract Back to Event Anti-thrombotic properties of a CD31-derived peptide coated on a surface Charlotte Rasser1, Frédéric Chaubet1, Caroline Jobin1, Giuseppina Caligiuri1 and Véronique Ollivier1 1 Laboratory for Vascular Translational Science - Inserm U1148, France Introduction: Thrombosis is a major concern in the design of most biomaterials, especially in those for vascular applications. Coating the biomaterial surface with a bioactive agent is one of the possible strategies to avoid thrombosis. CD31 (also known as PECAM-1) is a transmembrane glycoprotein present on endothelial cells, lymphocytes, neutrophils and platelets. CD31 has been demonstrated to inhibit platelet activation and thrombus formation[1],[2] and to modulate thrombin-induced tissue factor expression on endothelial cells[3].We therefore assessed the potential of a CD31-agonist peptide as an anti-thrombotic coating for biomaterials. Materials and Methods: The CD31-agonist peptide was left to adsorb on a 96-well plate with high binding affinity (Immulon® 2 HB, Thermo Scientific). Excess peptide was rinsed with phosphate buffered saline (PBS). Unmodified wells of the same plate were used as control. In a first set of experiments, thrombin generation in platelet-rich plasma (PRP) was performed using a Calibrated Automated Thrombogram (CAT) in presence of 0.5 pM tissue factor (TF), in a Fluoroskan Ascent (Thermo Scientific). In a second set, human coronary artery endothelial cells (HCAEC) were cultured in a 96-well plate with or without peptide coating until confluence was reached. Then, thrombin generation was measured at the surface of the cells in platelet-poor plasma (PPP) and in presence of 1 pM TF. To estimate cell viability, a tetrazolium (MTT, Sigma Aldrich) colorimetric assay was performed in parallel. Figure 1. Typical curve of thrombin generation in PRP in presence of 0.5 pM TF, in control wells or peptide-coated wells. n=6. Figure2. a) Typical curve of thrombin generation in PPP, in presence of 1 pM TF, at the surface of endothelial cells cultivated either in control wells or peptide-coated wells. b) MTT assay. n=3. Results and Discussion: Figure 1 presents the anti-platelet action of the peptide (thrombograms acquired in PRP, without endothelial cells). Thrombin generation is significantly reduced (-40%) in the peptide-coated wells, suggesting that the CD31-derived peptide is able to control the activation of the platelets coming into contact with the surface. In addition, thrombin generation at the surface of endothelial cells was reduced by 50% when the cells were cultured onto the peptide-coated wells (Figure 2a) as compared to unmodified wells. Since the number of endothelial cells in the two types of wells was not significantly different, as demonstrated by the MTT assay (Figure 2b), the peptide has most likely acted on the phenotype of the endothelial cells, promoting their anti-thrombotic phenotype. Conclusion: Our data show that coating surfaces with a CD31-agonist peptide results in a dual anti-thrombotic action: on the platelets coming into direct contact with the coated surface as well as on endothelial cells that shall grow on it. The CD31-derived peptide thus appears as a promising bioactive agent for biomaterial coating in applications such as vascular stents.

High-density lipoprotein from patients with coronary heart disease loses anti-thrombotic effects on endothelial cells: impact on arterial thrombus formation.

Thrombus formation is determined by the balance between pro- thrombotic mediators and anti-thrombotic factors.High-density lipoprotein (HDL) from healthy subjects exerts anti-thrombotic properties. Whether this is also the case for HDL from patients with stable coronary heart disease (CHD) or acute coronary syndrome (ACS) is unknown.In human aortic endothelial cells in culture,HDL (50 µg/ml) from healthy subjects (HS) inhibited thrombin-induced tissue factor (TF) expression and activity, while HDL (50 µg/ml) from CHD and ACS patients did not. Similarly, only healthy HDL increased endothelial tissue factor pathway inhibitor (TFPI) expression and tissue plasminogen activator (tPA) release, while HDL from CHD and ACS patients had no effect. Healthy HDL inhibited thrombin-induced plasminogen activator inhibitor type 1 (PAI-1) expression, while HDL from ACS patients enhanced endothelial PAI-1 expression. Inhibition of nitric oxide (NO) formation with L-NAME (100 µmol/l) abolished the anti-thrombotic effects of healthy HDL on TF, TFPI, and tPA expression. The exogenous nitric oxide donor, DETANO, mimicked the effects of healthy HDL and counterbalanced the loss of anti-thrombotic effects of HDL from CHD and ACS patients in endothelial cells. In line with this observation, healthy HDL, in contrast to HDL from CHD and ACS patients, increased endothelial NO production. In the laser-injured carotid artery of the mouse, thrombus formation was delayed in animals treated with healthy HDL compared with mice treated with vehicle or HDL from patients with CHD or ACS. In conclusion, HDL from CHD and ACS patients loses the ability of healthy HDL to suppress TF and to increase TFPI and t-PA and instead enhances PAI-1 and arterial thrombus formation.

Vascular Gas6 contributes to thrombogenesis and promotes tissue factor up-regulation after vessel injury in mice

AbstractGas6 (growth-arrest specific gene 6) plays a role in thrombus stabilization. Gas6 null (−/−) mice are protected from lethal venous and arterial thromboembolism through platelet signaling defects induced only by 5μM ADP and 10μM of the thromboxane analog, U46619. This subtle platelet defect, despite a dramatic clinical phenotype, raises the possibility that Gas6 from a source other than platelets contributes to thrombus formation. Thus, we hypothesize that Gas6 derived from the vascular wall plays a role in venous thrombus formation. Bone marrow transplantation and platelet depletion/reconstitution experiments generating mice with selective ablations of Gas6 from either the hematopoietic or nonhematopoietic compartments demonstrate an approximately equal contribution by Gas6 from both compartments to thrombus formation. Tissue factor expression was significantly reduced in the vascular wall of Gas6−/− mice compared with WT. In vitro, thrombin-induced tissue factor expression was reduced in Gas6−/− endothelial cells compared with wild-type endothelium. Taken together, these results demonstrate that vascular Gas6 contributes to thrombus formation in vivo and can be explained by the ability of Gas6 to promote tissue factor expression and activity. These findings support the notion that vascular wall-derived Gas6 may play a pathophysiologic role in venous thromboembolism.

Bivalirudin Inhibits Periprocedural Platelet Function and Tissue Factor Expression of Human Smooth Muscle Cells

A major concern of stent implantation after percutaneous coronary intervention (PCI) is acute stent thrombosis. Effective inhibition of periprocedural platelet function in patients with coronary artery disease (CAD) leads to an improved outcome. In this study, we examined the periprocedural platelet reactivity after administrating bivalirudin during PCI compared to unfractionated heparin (UFH) administration. Further, the effect of bivalirudin on induced tissue factor (TF) expression in smooth muscle cells (SMC) was determined. Patients with CAD (n = 58) and double antithrombotic medication were treated intraprocedural with UFH (n = 30) or bivalirudin (n = 28). Platelet activation markers were flow cytometrically measured before and after stenting. The expression of TF in SMC was determined by real-time PCR and Western blotting. The thrombogenicity of platelet-derived microparticles and SMC was assessed via a TF activity assay. Bivalirudin significantly diminished the agonist-induced platelet reactivity post-PCI. Compared to UFH treatment, the adenosine diphosphate (ADP) and thrombin receptor-activating peptide (TRAP)-induced thrombospondin expression post-PCI was reduced when bivalirudin was administrated during intervention. In contrast to UFH, bivalirudin reduced the P-selectin expression of unstimulated and ADP-induced platelets post-PCI. Moreover, bivalirudin inhibited the thrombin-, but not FVIIa- or FVIIa/FX-induced TF expression and pro-coagulant TF activity of SMC. Moreover, bivalirudin reduced the TF activity of platelet-derived microparticles postinduction with TRAP or ADP. Bivalirudin is better than UFH in reducing periprocedural platelet activation. Moreover, thrombin-induced TF expression is inhibited by bivalirudin. Thus, bivalirudin seems to be a better anticoagulant during PCI than UFH.

Peroxisome Proliferator-Activated Receptor-Gamma Agonists Suppress Tissue Factor Overexpression in Rat Balloon Injury Model with Paclitaxel Infusion

The role and underlying mechanisms of rosiglitazone, a peroxisome proliferator-activated receptor-gamma (PPAR-γ) agonist, on myocardial infarction are poorly understood. We investigated the effects of this PPAR-γ agonist on the expression of tissue factor (TF), a primary molecule for thrombosis, and elucidated its underlying mechanisms. The PPAR-γ agonist inhibited TF expression in response to TNF-α in human umbilical vein endothelial cells, human monocytic leukemia cell line, and human umbilical arterial smooth muscle cells. The overexpression of TF was mediated by increased phosphorylation of mitogen-activated protein kinase (MAPK), which was blocked by the PPAR-γ agonist. The effective MAPK differed depending on each cell type. Luciferase and ChIP assays showed that transcription factor, activator protein-1 (AP-1), was a pivotal target of the PPAR-γ agonist to lower TF transcription. Intriguingly, two main drugs for drug-eluting stent, paclitaxel or rapamycin, significantly exaggerated thrombin-induced TF expression, which was also effectively blocked by the PPAR-γ agonist in all cell types. This PPAR-γ agonist did not impair TF pathway inhibitor (TFPI) in three cell types. In rat balloon injury model (Sprague-Dawley rats, n = 10/group) with continuous paclitaxel infusion, the PPAR-γ agonist attenuated TF expression by 70±5% (n = 4; P<0.0001) in injured vasculature. Taken together, rosiglitazone reduced TF expression in three critical cell types involved in vascular thrombus formation via MAPK and AP-1 inhibitions. Also, this PPAR-γ agonist reversed the paclitaxel-induced aggravation of TF expression, which suggests a possibility that the benefits might outweigh its risks in a group of patients with paclitaxel-eluting stent implanted.

Atorvastatin neutralises the thrombin-induced tissue factor expresion in endothelial cells via geranylgeranyl pyrophosphate

Statins may have beneficial effects in atherogenesis given their antithrombotic properties involving non-lipid mechanisms that modify endothelial function of tissue factor induction by thrombin. In this study, we investigate the effect of atorvastatin on tissue factor (TF) activity in thrombin-stimulated endothelial cells and its regulation through mevalonate or its derivatives. First subculture of human umbilical endothelial cells was used for this study. Cells were treated with thrombin and atorvastatin for different time intervals and dosage. Tissue factor activity was measured as Factor Xa generation induced by Tissue Factor-Factor VIIa complex on confluent cells. Our results show that atorvastatin prevents the thrombin-induced up-regulation of tissue factor activity in a concentration-dependent manner. Mevalonate and geranylgeranyl pyrophosphate reversed this inhibitory effect of atorvastatin on tissue factor activity, while the presence of farnesyl pyrophosphate did not prevent the atorvastatin effect on thrombin-induced tissue factor activity. Rho-kinase inhibitor did not affect the thrombin stimulation of tissue factor activity. High amount of hydrophobic isoprenoid groups decreases the thrombin-induced TF activity and may promote endothelial cell anti-thrombotic action. Rho kinase pathways do not have a major role in the thrombin-mediated TF activity. The inhibitory effect of atorvastatin on thrombin-induced TF activity was partially reversed by MVA and GGPP but not FPP.

Opposing and uncoupling effects of mTOR and S6K1 in the regulation of endothelial tissue factor expression

Rapamycin has been reported to enhance tissue factor (TF) expression. The present study investigated roles of mammalian target of rapamycin (mTOR) and its downstream S6K1 in this process. We showed here that, consistent with rapamycin, knocking-down mTOR enhanced thrombin-induced TF mRNA and protein levels, whereas silencing S6K1 mitigated up-regulation of TF protein but not TF mRNA level. The enhanced TF protein level upon mTOR-silencing was further augmented by over-expression of a constitutively active S6K1 mutant and reduced by blocking RhoA, p38 mapk or NF-κB. The results reveal an opposing and uncoupling effect of mTOR and S6K1 in regulating TF expression.

Abstract 5486: High Density Lipoprotein (HDL) From Healthy Subjects, but Not From Patients With Coronary Artery Disease, Exerts Anti-thrombotic Effects on Human Endothelial Cells

Background: Arterial thrombus formation is determined by the balance between pro-thrombotic mediators such as tissue factor (TF) and plasminogen activator inhibitor type 1 (PAI-1), and anti-thrombotic factors like tissue factor pathway inhibitor (TFPI) or tissue plasminogen activator (tPA). Native HDL from healthy subjects (HS) has anti-thrombotic properties; however, it remains unknown whether this is the case for HDL from patients with stable coronary disease (CAD) or acute coronary syndrome (ACS). Methods: HDL was isolated by sequential ultracentrifugation from HS and patients with CAD and ACD. The effects of HDL (50 μ g/ml) on TF, TFPI, and PAI-1 expression in human endothelial cells were determined by Western blot analysis; tPA release was measured by ELISA. Results: HDL from HS impaired thrombin-induced TF expression (−45±5%, p&lt;0.05, n=16) and activity (−33±8%, p&lt;0.05, n±7); in contrast, HDL from CAD and ACS patients did not (p=NS, n=12 and n=8). Similarly, HDL from HS increased TFPI expression by 2-fold (p&lt;0.01, n=8), while HDL from CAD and ACS patients had no effect (p=NS). HDL from HS enhanced tPA release (+26±3%; p=0.05, n=8); in contrast, HDL from CAD and ACS patients did not (p=NS, n=6). Furthermore, HDL from HS did not affect PAI-1 expression, while HDL from CAD patients enhanced PAI-1 expression by 62% (p&lt;0.05 vs. healthy, n=12) and HDL from ACS patients by 2 fold (p&lt;0.05 vs. control and p&lt;0.05 vs. healthy, n=8). Pretreatment with the inhibitor of NO formation, L-NAME (100 μ M), abolished the anti-thrombotic effects of HDL from HS on TF, TFPI, and tPA expression. The exogenous nitric oxide donor, DETANO, mimicked the effects of HDL from HS on TF, TFPI, and tPA. Conclusion: This study demonstrates that HDL from healthy subjects exerts anti-thrombotic effects on endothelial cells. In contrast, HDL from CAD and ACS patients loses these antithrombotic properties and instead enhances PAI-1 expression, thereby becoming pro-thrombotic. This observation might be highly relevant for HDL-targeted therapies.

Amiodarone Inhibits Arterial Thrombus Formation and Tissue Factor Translation

In patients with coronary artery disease and reduced ejection fraction, amiodarone reduces mortality by decreasing sudden death. Because the latter may be triggered by coronary artery thrombosis as much as ventricular arrhythmias, amiodarone might interfere with tissue factor (TF) expression and thrombus formation. Clinically relevant plasma concentrations of amiodarone reduced TF activity and impaired carotid artery thrombus formation in a mouse photochemical injury model in vivo. PTT, aPTT, and tail bleeding time were not affected; platelet number was slightly decreased. In human endothelial and vascular smooth muscle cells, amiodarone inhibited tumor necrosis factor (TNF)-alpha and thrombin-induced TF expression as well as surface activity. Amiodarone lacking iodine and the main metabolite of amiodarone, N-monodesethylamiodarone, inhibited TF expression. Amiodarone did not affect mitogen-activated protein kinase activation, TF mRNA expression, and TF protein degradation. Metabolic labeling confirmed that amiodarone inhibited TF protein translation. Amiodarone impairs thrombus formation in vivo; in line with this, it inhibits TF protein expression and surface activity in human vascular cells. These pleiotropic actions occur within the range of amiodarone concentrations measured in patients, and thus may account at least in part for its beneficial effects in patients with coronary artery disease.

Plasmin Enhances Cell Surface Tissue Factor Activity without Increasing Tissue Factor Antigen Levels at the Cell Surfaces of Mesothelial and Endothelial Cells.

Mesothelial cells that line the thoracic and peritoneal cavities play an important role in protecting the heart, lungs and internal organs and keeping the surfaces free of friction and non-adhesive. Although human mesothelial cells in culture express low levels of tissue factor (TF), TF expression was not detected in vivo in the mesothelial lining of normal pleura but was detected in the mesothelium overlying injured or inflamed lung tissue. Inflammation in the lung parenchyma predisposes to the development of inflammatory exudative pleural effusions and pleural loculation, but the mechanism(s) responsible for TF expression in mesothelial cells under such conditions remains unclear. In the present study, we investigated whether plasmin and thrombin, two major proteases present in exudative pleural effusions, induce TF expression in cultured primary human pleural mesothelial cells (HMC). Confluent monolayers of HMC were treated with plasmin (50 nM) and thrombin (5 nM) for varying time periods. TF expression was analyzed by measurement of cell surface TF activity in FX activation assay, TF antigen by Western blotting and TF mRNA by Northern blot analysis. Both plasmin and thrombin increased cell surface TF activity by 3–4 fold. Thrombin markedly increased TF antigen levels. Surprisingly, only a minimal increase in TF antigen levels was seen in the plasmin-treated cells and no increase in TF mRNA was observed in these cells. Interestingly, 75% of the increase in TF activity was observed within 30 min of plasmin treatment. In thrombin-stimulated cells, TF activity increased slowly, reaching maximum at 4–6 h. Treatment of HMC with actinomycin-D or cycloheximide prior to the addition of the protease failed to inhibit the plasmin-induced TF activity whereas they completely attenuated the thrombin-induced TF activity. These data suggest that plasmin and thrombin enhance TF activity in HMC by different mechanisms, and that plasmin-induced TF activity is independent of de novo synthesis of TF. We also examined the effect of plasmin on TF activity in human umbilical vein endothelial cells (HUVEC). Although plasmin had a minimal effect on naive HUVEC, 30 min plasmin treatment increased cell surface TF activity of thrombin-stimulated endothelial cells by more than two-fold. In our earlier studies, we showed that proteases could mobilize intracellular TF to the cell surface of fibroblasts and thus enhance TF activity independent of de novo synthesis of TF. However, FACS analysis and 125I-FVIIa binding to cell surface TF revealed no significant differences in TF antigen levels at the surfaces of control and plasmin-stimulated HMC. Since exposure of anionic phospholipids at the outer cell surface membrane can increase cell surface TF activity, we next sought to determine if plasmin increases anionic phospholipids on the outer bilayer of the HMC cell membrane. Prothrombin activation assays showed no increase in anionic phospholipids at the surface in plasmin-treated cells. Although recent studies suggest that disulfide bond formation of cysteines in TF enhances TF procoagulant activity, our recent studies in other cell model systems suggest that this mechanism is unlikely to account for plasmin-mediated induction of cell surface TF activity in HMC. While further studies are required to exclude this possibility, our present data indicate that plasmin enhances cell surface TF activity of HMC and HUVEC by a novel mechanism that is yet to be elucidated.

Histamine differentially interacts with tumor necrosis factor-alpha and thrombin in endothelial tissue factor induction: the role of c-Jun NH2-terminal kinase.

Histamine plays an important role in vascular disease. Tissue factor (TF) expression is induced in vascular inflammation and acute coronary syndromes. This study examined the effect of histamine on tumor necrosis factor-alpha- (TNF-alpha-) vs. thrombin-induced endothelial TF expression. Histamine (10(-8)-10(-5) mol L-1), TNF-alpha (5 ng mL-1), and thrombin (1 U mL-1) induced TF expression in human endothelial cells. Although TF expression by TNF-alpha and thrombin was identical, histamine augmented TNF-alpha-induced expression 7.0-fold, but thrombin-induced expression only 2.6-fold. Similar responses occurred with TF activity. The H1-receptor antagonist mepyramine abrogated these effects. Differential augmentation by histamine was also observed at the mRNA level. Histamine-induced p38 activation preceded a weak second activation to both TNF-alpha and thrombin. Histamine-induced c-Jun NH2-terminal kinase (JNK) activation was followed by a strong second activation to TNF-alpha, and less to thrombin. Selective inhibition of this second JNK activation by SP600125 reduced TF induction to histamine plus TNF-alpha by 67%, but to histamine plus thrombin by only 32%. Histamine augmented TNF-alpha- and thrombin-induced vascular cell adhesion molecule 1 (VCAM-1) expression to a similar extent. Consistent with this observation, VCAM-1 induction to TNF-alpha and thrombin was mediated by p38, but not by JNK. Histamine differentially augments TNF-alpha- vs. thrombin-induced TF expression and activity, which is mediated by the H1-receptor, occurs at the mRNA level, and is related to differential JNK activation.

Paclitaxel Enhances Thrombin-Induced Endothelial Tissue Factor Expression via c-Jun Terminal NH 2 Kinase Activation

Paclitaxel is used on drug-eluting stents because it inhibits proliferation of vascular cells. Stent thrombosis remains a concern with this compound, particularly with higher dosages. This study investigates the effect of paclitaxel on tissue factor (TF) expression in human endothelial cells. Paclitaxel enhanced thrombin-induced endothelial TF protein expression in a concentration- and time-dependent manner. A concentration of 10(-5) mol/L elicited a 2.1-fold increase in TF protein and a 1.6-fold increase in TF surface activity. The effect was similar after a 1 hour as compared with a 25-hour pretreatment period. Real-time polymerase chain reaction revealed that paclitaxel increased thrombin-induced TF mRNA expression. Paclitaxel potently activated c-Jun terminal NH2 kinase (JNK) as compared with thrombin alone, whereas the thrombin-mediated phosphorylation of p38 and extracellular signal-regulated kinase remained unaffected. Similar to paclitaxel, docetaxel enhanced both TF expression and JNK activation as compared with thrombin alone. The JNK inhibitor SP600125 reduced thrombin-induced TF expression by 35%. Moreover, SP600125 blunted the effect of paclitaxel and docetaxel on thrombin-induced TF expression. Paclitaxel increases endothelial TF expression via its stabilizing effect on microtubules and selective activation of JNK. This observation provides novel insights into the pathogenesis of thrombus formation after paclitaxel-eluting stent deployment and may have an impact on drug-eluting stent design.

Cocaine unbalances endothelial tissue factor and tissue factor pathway inhibitor expression

Cocaine consumption can lead to myocardial infarction. Tissue factor (TF) has been implicated in acute coronary syndromes, and the balance of TF and tissue factor pathway inhibitor (TFPI) determines initiation of thrombus formation. This study was designed to investigate the effect of cocaine on endothelial TF and TFPI expression. Cocaine (10(-8)-10(-5) mol/l) increased thrombin-induced TF expression by 24% at 10(-7) mol/l (P < 0.001) without affecting basal TF expression. In contrast, cocaine reduced endothelial TFPI expression by 47% at 10(-7) mol/l (P < 0.01). Moreover, thrombin impaired endothelial TFPI expression, and cocaine (10(-8) mol/l) further reduced TFPI expression by 33% as compared to thrombin (P < 0.02). These effects occur at cocaine concentrations usually present in plasma of consumers. Given the importance of TF in the pathogenesis of acute coronary syndromes, TF induction in conjunction with TFPI suppression may be relevant for the increased frequency of myocardial infarction observed in cocaine consumers.

Rapamycin, but Not FK-506, Increases Endothelial Tissue Factor Expression

Drugs released from stents affect the biology of vascular cells. We examined the effect of rapamycin and FK-506 on tissue factor (TF) expression in human aortic endothelial cells (HAECs) and vascular smooth muscle cells (HAVSMCs). Rapamycin enhanced thrombin- and tumor necrosis factor (TNF)-alpha-induced endothelial TF expression in a concentration-dependent manner. The maximal increase was 2.5-fold more pronounced than that by thrombin or TNF-alpha alone and was paralleled by a 1.4-fold higher TF surface activity compared with thrombin alone. Rapamycin by itself increased basal TF levels by 40%. In HAVSMCs, rapamycin did not affect thrombin- or TNF-alpha-induced TF expression. In contrast to rapamycin, FK-506 did not enhance thrombin- or TNF-alpha-induced endothelial TF expression. Thrombin induced a transient dephosphorylation of the mammalian target of rapamycin downstream target p70S6 kinase. Rapamycin completely abrogated p70S6 kinase phosphorylation, but FK-506 did not. FK-506 antagonized the effect of rapamycin on thrombin-induced TF expression. Rapamycin did not alter the pattern of p38, extracellular signal-regulated kinase, or c-Jun NH2-terminal kinase phosphorylation. Real-time polymerase chain reaction analysis revealed that rapamycin had no influence on thrombin-induced TF mRNA levels for up to 2 hours but led to an additional increase after 3 and 5 hours. Rapamycin, but not FK-506, enhances TF expression in HAECs but not in HAVSMCs. This effect requires binding to FK binding protein-12, is mediated through inhibition of the mammalian target of rapamycin, and partly occurs at the posttranscriptional level. These findings may be clinically relevant for patients receiving drug-eluting stents, particularly when antithrombotic drugs are withdrawn or ineffective, and may open novel perspectives for the design of such stents.

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.

Thrombin activates the p21-activated kinase in pulmonary artery smooth muscle cells

The p21-activated serine/threonine kinases (PAK) play an important role in a variety of cellular functions. However, their role in the smooth muscle response to thrombin, which is activated upon vascular injury and promotes vascular remodelling processes, is not resolved. Here we investigated the role of PAK in thrombin signalling and regulation of tissue factor (TF), the activator of the extrinsic coagulation cascade, in pulmonary artery smooth muscle cells (PASMC), the main cell type responsible for vascular remodelling in pulmonary hypertension. PAK was rapidly phosphorylated in response to thrombin. Thrombin and active PAKT423E phosphorylated p38 MAP kinase (p38MAPK), ERK1/2, phosphatidylinositol-dependent kinase-1 (PDK1) and protein kinase B/Akt (PKB) whereas kinase-deficient PAK1 prevented activation of these kinases by thrombin. In addition, kinase- deficient MKK3 inhibited activation of PDK1 and PKB by thrombin. Further, thrombin and active PAK1 induced TF expression and promoter activity while kinase-deficient PAK1 diminished thrombin-induced TF upregulation. Moreover, kinase-deficient MKK3, PDK1 and PKB inhibited thrombin- and PAK-dependent TF expression and promoter activity. Together these findings show that PAK is a critical element of thrombin signalling in PASMC which is involved in the regulation of TF expression by sequentially activating MKK3/p38MAPK, PDK1 and PKB. Thus, PAK may play an important role in promoting vascular remodelling processes in pulmonary hypertension.

Rac regulates thrombin-induced tissue factor expression in pulmonary artery smooth muscle cells involving the nuclear factor-kappaB pathway.

Pulmonary hypertension is associated with enhanced thrombogenicity of the vessel wall contributing to vascular remodeling. However, the signaling mechanisms promoting this prothrombotic state are not resolved. Here we investigated the role of the GTPase Rac in the regulation of tissue factor (TF) expression and activity in response to thrombin in pulmonary artery smooth muscle cells (PASMC). TF mRNA and protein expression and surface procoagulant activity were increased by thrombin in PASMC. These responses were enhanced in the presence of the constitutively active Rac mutant RacG12V, but were abrogated in cells expressing dominant-negative RacT17N. Thrombin and RacG12V also increased human TF promoter activity primarily involving a sequence between -636 and -111 bp containing a distal, nuclear factor-kappaB (NFkappaB)-dependent enhancer element. Indeed, thrombin and RacG12V stimulated NFkappaB-dependent transcriptional activity, and overexpression of p50/p65 significantly increased human TF promoter activity. Moreover, in RacG12V-overexpressing cells, TF promoter activity was significantly decreased by coexpression of dominant-negative mutants of IkappaBalpha and IkappaBKalpha, which prevent NFkappaB activation. As enhanced NFkappaB activity has been observed in patients with pulmonary hypertension, Rac-dependent activation of the NFkappaB pathway may be a critical element promoting thrombin-induced TF expression and activity, and thus a prothrombotic state in pulmonary hypertension.

Synergistic effect of sphingosine 1-phosphate on thrombin-induced tissue factor expression in endothelial cells

AbstractSphingosine 1-phosphate (S1P), a bioactive lipid, is produced and stored in platelets and is released from activated platelets during blood coagulation activation. Thrombin, which is also generated during blood coagulation, has been shown to induce tissue factor (TF), the initiator of blood coagulation, in endothelial cells (ECs); however, the effect of S1P on this process is not evaluated. Here we demonstrated that S1P strongly potentiated thrombin-induced TF expression in ECs and that S1P itself did not induce TF expression. Among signaling lipids, platelet-activating factor slightly enhanced thrombin-induced TF expression; other lipids, including lysophosphatidic acid, lysophosphatidylcholine, sphingosine, and C2-ceramide exert no effect on TF expression. S1P enhanced TF expression at the transcriptional level, possibly via promoting the activation of transcription factors nuclear factor–κB (NF-κB) and Egr-1. Thrombin weakly and S1P strongly activated extracellular signal–regulated kinase 1/2 (ERK1/2) mitogen-activated protein (MAP) kinase and, in the presence of both stimulants, enhanced and sustained activation of this kinase was observed. The ERK1/2-specific inhibitor PD98059 significantly inhibited enhanced TF expression induced by both stimulants but only weakly inhibited thrombin-induced TF expression, thus indicating the requirement of the ERK1/2 pathway in synergistic induction of TF expression. In addition, we found that thrombin and S1P rapidly up-regulated the expression of S1P receptors, endothelial differentiation gene-1 (EDG-1) and EDG-3, thereby suggesting that the effect of S1P on TF expression and other EC functions may be enhanced by thrombin and S1P itself. The present data reveal the synergistic effect of S1P on thrombin-induced TF expression in ECs, which may promote further thrombin and S1P generation, thus propagating a positive feedback reaction.

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.