Initiator Of Blood Coagulation Research Articles

Abstract 491: Protein Kinase D2 Mediates LPA-induced Tissue Factor Expression and Activity through the MAPK Pathway in Mouse Aortic Smooth Muscle Cells

Lysophosphatidic acid (LPA), a potent bioactive lipid in oxidized-LDL, modulates vascular cell functions in vitro and in vivo via regulating the expression of specific functional genes. We previously reported that LPA highly induces the expression of tissue factor (TF) in vascular smooth muscle cells. TF is the principal initiator of blood coagulation and is important for thrombosis. However, the intracellular signaling pathway leading to LPA induction of TF is not well understood. In the current study, we found that LPA markedly induced protein kinase D (PKD) activation in mouse primary aortic smooth muscle cells (MASMCs). PKD consists of three isoforms (PKD1, PKD2 and PKD3), which belong to a family of serine/threonine protein kinases. We observed that all three PKD isoforms were expressed in MASMCs. LPA-induced PKD phosphorylation is via a PKC-dependent pathway. Employing a small-interfering RNA approach to knock down the expression of the specific isoforms of PKD in MASMCs, we found that knockdown of PKD2 expression blocked LPA-induced TF expression and activity, indicating that PKD2 is the key intracellular mediator for the expression and surface activity of TF. The role of PKD2 was further confirmed by overexpression of the dominant-negative PKD2. We observed that overexpression of the dominant-negative isoform of PKD2 blocked LPA-induced TF expression. Our data also demonstrate that PKD2 mediated LPA-induced TF expression via the mitogen-activated protein kinase pathway. Using primary MASMCs isolated from LPA receptor knockout mice, our data revealed that LPA receptor 1 was responsible for LPA-induced TF expression. Therefore, these data provide the first evidence that PKD is a new component in LPA signaling in vascular SMCs, leading to gene expression, as evidenced by TF expression. Our data provide new insights into the mechanisms of coagulation and possible therapeutic targets for atherothrombosis.

Disulfide reduction abolishes tissue factor cofactor function

BackgroundTissue factor (TF), an in vivo initiator of blood coagulation, is a transmembrane protein and has two disulfides in the extracellular domain. The integrity of one cysteine pair, Cys186–Cys209, has been hypothesized to be essential for an allosteric “decryption” phenomenon, presumably regulating TF procoagulant function, which has been the subject of a lengthy debate. The conclusions of published studies on this subject are based on indirect evidences obtained by the use of reagents with potentially oxidizing/reducing properties. MethodsThe status of disulfides in recombinant TF1–263 and natural placental TF in their non-reduced native and reduced forms was determined by mass-spectrometry. Functional assays were performed to assess TF cofactor function. ResultsIn native proteins, all four cysteines of the extracellular domain of TF are oxidized. Reduced TF retains factor VIIa binding capacity but completely loses the cofactor function. ConclusionThe reduction of TF disulfides (with or without alkylation) eliminates TF regulation of factor VIIa catalytic function in both membrane dependent FX activation and membrane independent synthetic substrate hydrolysis. General significanceResults of this study advance our knowledge on TF structure/function relationships.

Comparison of natural and recombinant tissue factor proteins: new insights

Tissue factor (TF), an initiator of blood coagulation in vivo, is expressed in a variety of cells. Sufficient natural TF has been isolated to clone and express recombinant proteins ranging from full-length TF to its extracellular domain. Because of the limited availability of natural TF, recombinant proteins have been used as surrogates. Despite the differences in their post-translational modifications, it has been accepted that membrane-anchored recombinant TFs are quite similar to the natural TF. Recent studies, however, have shown that post-translational modifications play an important role in TF-triggered thrombin generation.

Tissue Factor De-encryption, Thrombus Formation, and Thiol-disulfide Exchange

Tissue factor (TF) by forming a complex with factor VIIa (FVIIa) initiates blood coagulation. It was traditionally believed that the separation of FVIIa in circulation from subendothelial TF was the main control that was preventing spontaneous initiation of thrombosis and that circulating cells and endothelium did not express TF protein at rest in healthy individuals. However, TF has been detected in healthy human plasma and animal models of thrombosis, which indicate that TF in circulation can contribute to thrombin generation and fibrin formation after an activation event. Circulating TF-and indeed, most of the TF on the cell surface-is "encrypted" or coagulation inactive. The de-encryption step involves exposure of phosphatidylserine (PS), but PS exposure alone is insufficient for full TF activity. Allosteric disulfide bonds control protein function by mediating conformal change through the formation and breaking of disulfide bonds. TF contains a typical surface exposed allosteric bond in the membrane proximal fibronectin type III domain. Thiol-disulfide exchange involving this disulfide is implicated in TF activation with the formation of the disulfide bond corresponding with the active conformation of TF and free thiol or thiol-modified forms corresponding with encryption. Although the exact mechanism by which TF de-encryption occurs remains a subject of debate, thiol blockade and inhibition of oxidoreductases show an important role for thiol-disulfide reactions in platelet-independent pathways of coagulation in vitro and in vivo. In particular, redox active extracellular protein disulfide isomerase is involved in the earliest stages of thrombus initiation and has proven to be a potential target for antithrombotic drug development.

Tissue Factor as a Novel Target for Treatment of Breast Cancer

Tissue factor (TF), a 47-kDa transmembrane glycoprotein that initiates blood coagulation when complexed with factor VIIa (FVIIa), is expressed in several tumor types. TF has been shown to play a role in cell signaling, inflammation, angiogenesis, as well as tumor growth and metastasis. Activation of the TF signaling pathway has been implicated in mediating the function of many tumor cell types and has led to TF as a potential target in the treatment of several malignancies. Formation of the TF-FVIIa complex in breast cancer cells has been shown to exert an antiapoptotic effect and play a key role in tumor growth and metastasis. Breast cancer growth is suppressed by inhibition of TF-mediated PAR2 signaling, and deficiency in PAR2 delays spontaneous breast cancer development in mice. TF is expressed in triple-negative breast cancer (TNBC), an aggressive type of breast cancer in which there is currently a paucity of available targets. Various methods of targeting TF have been investigated and include immunoconjugates or icons, anti-TF antibodies, TF pathway inhibitors, targeted photodynamic therapy, and microRNAs. These investigations may give way to promising clinical therapies for breast cancer, especially in TNBC, for which there are relatively few effective treatment options.

Interferon-gamma–mediated tissue factor expression contributes to T-cell-mediated hepatitis through induction of hypercoagulation in mice

Concanavalin A (Con A) treatment induces severe hepatitis in mice in a manner dependent on T cells, interferon (IFN)-gamma, and tumor necrosis factor (TNF). Treatment with the anticoagulant heparin protects against hepatitis, despite healthy production of IFN-γ and TNF. Here, we investigated molecular and cellular mechanisms for hypercoagulation-mediated hepatitis. After Con A challenge, liver of wild-type (WT) mice showed prompt induction of Ifnγ and Tnf, followed by messenger RNA expression of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1), which initiate blood coagulation and inhibit clot lysis, respectively. Mice developed dense intrahepatic fibrin deposition and massive liver necrosis. In contrast, Ifnγ(-/-) mice and Ifnγ(-/-) Tnf(-/-) mice neither induced Pai1 or Tf nor developed hepatitis. In WT mice TF blockade with an anti-TF monoclonal antibody protected against Con A-induced hepatitis, whereas Pai1(-/-) mice were not protected. Both hepatic macrophages and sinusoidal endothelial cells (ECs) expressed Tf after Con A challenge. Macrophage-depleted WT mice reconstituted with hematopoietic cells, including macrophages deficient in signal transducer and activator of transcription-1 (STAT1) essential for IFN-γ signaling, exhibited substantial reduction of hepatic Tf and of liver injuries. This was also true for macrophage-depleted Stat1(-/-) mice reconstituted with WT macrophages. Exogenous IFN-γ and TNF rendered T-cell-null, Con A-resistant mice deficient in recombination-activating gene 2, highly susceptible to Con A-induced liver injury involving TF. Collectively, these results strongly suggest that proinflammatory signals elicited by IFN-γ, TNF, and Con A in both hepatic macrophages and sinusoidal ECs are necessary and sufficient for the development of hypercoagulation-mediated hepatitis.

Change of the initiation time of blood coagulation in pregnancy from 10-months to postpartum

We measured the time of initiation of blood coagulation (Ti) from pregnancy 10-months (36~40 weeks) till 1-month after delivery, paying particular attention to the very early postpartum period, using a damped oscillation rheometer that is approximately 160 times more sensitive than the Thromboelastogram® to evaluate the risk of thrombus formation. Blood samples were obtained from healthy volunteers at pregnancy 10-month, 1-hour, 3-hours, 4-days, 7-days, 3-weeks and 1-month after delivery. Ti values at pregnancy 10-month, 1-hour, 3-hours, 4-days, 7-days, 3-weeks, 1-month after delivery and in non-pregnant females were 20.4 ± 2.2, 11.7 ± 1.6, 13.2 ± 3.1, 17.2 ± 2.0, 20.2 ± 1.6, 21.4 ± 4.0, 24.6 ± 3.6, and (25.0 ± 3.4) minutes, respectively. Ti was significantly shorter at pregnancy 10-month, 1-hour, 3-ours, 4-days, 7-days and 3-weeks after delivery than in non-pregnant females. These data show that the blood of pregnant females is more hypercoagulable than non-pregnant females from pregnancy 10-month until 3-weeks post delivery, suggesting that they are at high risk of VTE after discharge from hospital.

Divergent Effect of Insulin On Whole-Blood Tissue Factor Procoagulant Activity in the Presence and Absence of Lipopolysaccharide

Abstract 636 Background:Individuals with diabetes mellitus (DM) have accelerated atherosclerosis and are prone to acute vascular events. Both hyperglycemia (HG) and hyperinsulinemia (HI) are independent risk factors for mortality in Type 2 DM (T2DM). Tissue factor (TF) is the principal initiator of blood coagulation. There is a circulating pool of TF in blood, which is thrombogenic. We have shown that membrane-bound TF levels are elevated in T2DM (J Clin Endocrinol Metab 2007, 92; 4352–8), and T1DM (Diabetes Care, 2012;35:1322–7). Infusion studies in normal healthy subjects showed that HG (∼200 mg/dl), HI and combined HG+HI elevated whole blood TF-procoagulant activity (TF-PCA) by 2, 6 and 9-fold, respectively (Diabetes 2006, 55; 202–8). In T2DM patients, the elevation by HI was 30%, and by HG+HI was 80% (J Clin Endocrinol Metab 2007, 92; 4352–8). However, in contrast to the findings in T2DM, our studies in T1DM patients (Diabetes Care, 2012;35:1322–7) showed that neither acutely raising blood glucose or insulin for 24 h, nor raising blood glucose together with insulin for up to 24 h increased TF-PCA. In fact, TF-PCA levels decreased during combined HG and HI and selective HG. To better understand the roles of HG and HI on TF-PCA, we studied their effects in vitro in whole blood from healthy individuals under both unstimulated and lipopolysaccharide (LPS)-stimulated conditions; the latter was done because patients with T2DM have evidence of platelet/monocyte activation as well as elevated plasma LPS levels. Methods:Glucose (200 mg/dL, HG) and insulin (1, 10 or 100 nM, HI) were added either alone or in combination (HG+HI) to blood from 6 healthy subjects and incubated for 4 hr at 37°C. These studies were performed in the presence or absence of 1 mg/ml LPS. Membrane bound TF-PCA was measured in whole blood lysates by a two-stage clotting assay (Key et al, Blood 1998, 91; 4216–23). Results:Basal glucose and insulin levels were 75±4 mg/dL and 99±24 pmol/L (n=6, mean±SE), respectively. With added glucose (HG) TF-PCA increased from baseline (22±5 U/ml mean ± SE) to 41±7 U/ml (p=0.006) at 2 hr and to 57±10 U/ml (p=0.0005) at 4 hr. Similarly, at 2 hr there was an increase with insulin 10 nM (33±8, p=0.033) and 100 nM (44±12, p=0.04). A combination of HG with increasing insulin concentrations (1, 10 or 100 nM) increased TF-PCA at 2 hr to 42±6 (p=0.04), 50±9 (p=0.02), and 63±12 (p=0.002), respectively. At 4 hr the corresponding levels were 71±16 (p=0.031), 69±15 (p=0.003), and 93±20 (p=0.004), respectively. Thus, HG, HI and HG+HI increased TF-PCA levels with highest levels being with the combination. This is in line with the findings from our infusion studies in healthy subjects. Studies in the presence of LPS:LPS alone induced a marked increase in TF-PCA from 22±5 U/ml to 826±93 (p<0.001) by 2 hr and to 591±113 (p<0.01) by 4 hr. The small increase with HG in presence of LPS was not statistically significant. With added insulin TF-PCA at 2 hr was lower than with LPS alone (826±93 U/ml) with 10 nM insulin (587±119, p=0.037) and 100 nM insulin (552±207, p=0.025). This was noted at 4 hr with 10 nM insulin (591±113 vs 472±83, p=0.014). This is in contrast to the findings in the absence of LPS-stimulation where HI stimulated TF-PCA. Notably, in the presence of LPS, the combination of HG with increasing HI caused a marked increase in TF-PCA at 2 hr over that with LPS alone (826±93 U/ml) at 1 nM insulin (2369±234, p=0.003) and 100 nM insulin (1992±288, p=0.009). This persisted at 4 hr (LPS: 591±113 vs 2595±49, p=0.006; and vs 1936±347, p= 0.005). TF-PCA with HG + HI at 2 and 4 hr was higher than with LPS plus glucose or insulin alone. Conclusions:In non-diabetic subjects HG and HI individually and in combination, increased whole blood TF-PCA. However, insulin inhibited the marked increase in TF-PCA induced by LPS. These findings suggest that the insulin effect is influenced by the cellular activation state. This may be relevant because platelets/monocytes are activated and endogenous LPS levels are elevated in DM. In LPS-stimulated samples, combined HG + HI induced the highest TF-PCA levels suggesting that HG may override the inhibitory effect of insulin in the presence of LPS. Further studies, including in patients with T1DM and T2DM, are needed to understand the effects of HG and HI and the cellular activation state on TF expression. Disclosures:No relevant conflicts of interest to declare.

Effect of Zymogen Domains and Active Site Occupation on Activation of Prothrombin by von Willebrand Factor-binding Protein

Prothrombin is conformationally activated by von Willebrand factor-binding protein (vWbp) from Staphylococcus aureus through insertion of the NH(2)-terminal residues of vWbp into the prothrombin catalytic domain. The rate of prothrombin activation by vWbp(1-263) is controlled by a hysteretic kinetic mechanism initiated by substrate binding. The present study evaluates activation of prothrombin by full-length vWbp(1-474) through activity progress curve analysis. Additional interactions from the COOH-terminal half of vWbp(1-474) strengthened the initial binding of vWbp to prothrombin, resulting in higher activity and an ∼100-fold enhancement in affinity. The affinities of vWbp(1-263) or vWbp(1-474) were compared by equilibrium binding to the prothrombin derivatives prethrombin 1, prethrombin 2, thrombin, meizothrombin, and meizothrombin(des-fragment 1) and their corresponding active site-blocked analogs. Loss of fragment 1 in prethrombin 1 enhanced affinity for both vWbp(1-263) and vWbp(1-474), with a 30-45% increase in Gibbs free energy, implicating a regulatory role for fragment 1 in the activation mechanism. Active site labeling of all prothrombin derivatives with D-Phe-Pro-Arg-chloromethyl ketone, analogous to irreversible binding of a substrate, decreased their K(D) values for vWbp into the subnanomolar range, reflecting the dependence of the activating conformational change on substrate binding. The results suggest a role for prothrombin domains in the pathophysiological activation of prothrombin by vWbp, and may reveal a function for autocatalysis of the vWbp·prothrombin complexes during initiation of blood coagulation.

Histamine induces activation of protein kinase D that mediates tissue factor expression and activity in human aortic smooth muscle cells

Histamine, an inflammatory mediator, has been shown to influence the pathogenesis of vascular wall cells. However, the molecular basis of its influence is not well understood. Our data reveal that histamine markedly induces protein kinase D (PKD) activation in human aortic smooth muscle cells. PKD belongs to a family of serine/threonine protein kinases, and its function in vascular disease is largely unknown. Our data show that histamine-induced PKD phosphorylation is dependent on the activation of histamine receptor 1 and protein kinase C (PKC). To determine the role of PKD in the histamine pathway, we employed a small-interfering RNA approach to downregulate PKD expression and found that PKD1 and PKD2 are key mediators for expression of tissue factor (TF), which is the key initiator of blood coagulation and is important for thrombosis. Our results show that PKD2 predominantly mediates histamine-induced TF expression via the p38 mitogen-activated protein kinase (MAPK) pathway, whereas PKD1 mediates histamine-induced TF expression through a p38 MAPK-independent pathway. We demonstrate that histamine induces TF expression via the PKC-dependent PKD activation. Our data provide the first evidence that PKD is a new component in histamine signaling in live cells and that PKD has a novel function in the histamine signaling pathway leading to gene expression, as evidenced by TF expression. Importantly, our data reveal a regulatory link from histamine to PKD and TF, providing new insights into the mechanisms of coagulation and the development of atherothrombosis.

Ets-1 transcription is required in tissue factor driven microvessel formation and stabilization

Tissue factor (TF) has well-recognized roles as initiator of blood coagulation as well as an intracellular signaling receptor. TF signaling regulates gene transcription and protein translation. Recently, we have shown that TF-induced mature neovessel formation is ultimately driven by CCL2 expression. However, the signaling process induced by TF to promote microvessel formation remains to be determined. This study was designed with the objective to investigate the mechanisms involved in TF-induced neovessel formation. Here, we have identified that Ets-1 expression is a downstream effector of TF signaling. TF-siRNA induced a highly significant reduction in Ets-1 expression levels and in Ets-1/DNA binding while inducing abrogation of microvessel formation. Activation of Ets-1 rescued the effect of TF inhibition and restored microvessel formation confirming the critical role of Ets-1 in TF-induced angiogenesis. VE-cadherin expression, a key regulator of endothelial intercellular junctions, and an Ets-1 target molecule was dependent of TF-inhibition. We show that TF signals through ERK1/2 to activate Ets-1 and induce CCL2 gene expression by binding to its promoter region. We conclude that endothelial cell TF signals through ERK1/2 and Ets-1 to trigger microvessel formation.

Cyclooxygenase-2–Derived Prostacyclin Regulates Arterial Thrombus Formation by Suppressing Tissue Factor in a Sirtuin-1–Dependent-Manner

Selective inhibitors of cyclooxygenase (COX)-2 increase the risk of myocardial infarction and thrombotic events, but the responsible mechanisms are not fully understood. We found that ferric chloride-induced arterial thrombus formation was significantly greater in COX-2 knockout compared with wild-type mice. Cross-transfusion experiments excluded the likelihood that COX-2 knockout platelets, despite enhanced aggregation responses to collagen and thrombin, are responsible for increased arterial thrombus formation in COX-2 knockout mice. Importantly, we observed that COX-2 deletion decreased prostacyclin synthase and production and peroxisome proliferator-activated receptor- and sirtuin-1 (SIRT1) expression, with consequent increased upregulation of tissue factor (TF), the primary initiator of blood coagulation. Treatment of wild-type mice with a prostacyclin receptor antagonist or a peroxisome proliferator-activated receptor-δ antagonist, which predisposes to arterial thrombosis, decreased SIRT1 expression and increased TF activity. Conversely, exogenous prostacyclin or peroxisome proliferator-activated receptor-δ agonist completely reversed the thrombotic phenotype in COX-2 knockout mice, restoring normal SIRT1 levels and reducing TF activity. Furthermore, inhibition of SIRT1 increased TF expression and activity and promoted generation of occlusive thrombi in wild-type mice, whereas SIRT1 activation was sufficient to decrease abnormal TF activity and prothrombotic status in COX-2 knockout mice. Modulation of SIRT1 and hence TF by prostacyclin/peroxisome proliferator-activated receptor-δ pathways not only represents a new mechanism in controlling arterial thrombus formation but also might be a useful target for therapeutic intervention in the atherothrombotic complications associated with COX-2 inhibitors.

Upregulation of the Coagulation Factor VII Gene during Glucose Deprivation Is Mediated by Activating Transcription Factor 4

BackgroundConstitutive production of blood coagulation proteins by hepatocytes is necessary for hemostasis. Stressful conditions trigger adaptive cellular responses and delay processing of most proteins, potentially affecting plasma levels of proteins secreted exclusively by hepatocytes. We examined the effect of glucose deprivation on expression of coagulation proteins by the human hepatoma cell line, HepG2.Methodology/Principal FindingsExpression of coagulation factor VII, which is required for initiation of blood coagulation, was elevated by glucose deprivation, while expression of other coagulation proteins decreased. Realtime PCR and ELISA demonstrated that the relative percentage expression +/− SD of steady-state F7 mRNA and secreted factor VII antigen were significantly increased (from 100+/−15% to 188+/−27% and 100+/−8.8% to 176.3+/−17.3% respectively, p<0.001) at 24 hr of treatment. The integrated stress response was induced, as indicated by upregulation of transcription factor ATF4 and of additional stress-responsive genes. Small interfering RNAs directed against ATF4 potently reduced basal F7 expression, and prevented F7 upregulation by glucose deprivation. The response of the endogenous F7 gene was replicated in reporter gene assays, which further indicated that ATF4 effects were mediated via interaction with an amino acid response element in the F7 promoter.Conclusions/SignificanceOur data indicated that glucose deprivation enhanced F7 expression in a mechanism reliant on prior ATF4 upregulation primarily due to increased transcription from the ATF4 gene. Of five coagulation protein genes examined, only F7 was upregulated, suggesting that its functions may be important in a systemic response to glucose deprivation stress.

Tissue factor–bearing exosome secretion from human mechanically stimulated bronchial epithelial cells in vitro and in vivo

Tissue factor (TF), a primary initiator of blood coagulation, also plays a pivotal role in angiogenesis. TF expression in the airways is associated with asthma, a disease characterized in part by subepithelial angiogenesis. To determine potential sources of TF and the mechanisms of its availability in the lung microenvironment. Normal human bronchial epithelial cells grown in air-liquid interface culture were subjected to a compressive stress of 30 cm H(2)O; this is comparable to that generated in the airway epithelium during bronchoconstriction in asthma. Conditioned media and cells were harvested to measure TF mRNA and TF protein. We also tested bronchoalveolar lavage fluid and airway biopsies from asthmatic patients and healthy controls for TF. TF mRNA was upregulated 2.2-fold after 3 hours of stress compared with unstressed cells. Intracellular and secreted TF proteins were enhanced 1.6-fold and more than 50-fold, respectively, compared with those of control cells after the onset of compression. The amount of TF in the bronchoalveolar lavage fluid from patients with asthma was found at mean concentrations that were 5 times greater than those of healthy controls. Immunohistochemical staining of endobronchial biopsies identified epithelial localization of TF with increased expression in asthma. Exosomes isolated from the conditioned media of normal human bronchial epithelial cells and the bronchoalveolar lavage fluid of asthmatic subjects by ultracentrifugation contained TF. Our invitro and invivo studies show that mechanically stressed bronchial epithelial cells are a source ofsecreted TF and that exosomes are potentially a key carrierofthe TF signal.

Opposite regulation by PI3K/Akt and MAPK/ERK pathways of tissue factor expression, cell-associated procoagulant activity and invasiveness in MDA-MB-231 cells

BackgroundTissue factor (TF), an initiator of blood coagulation, participates in cancer progression and metastasis. We recently found that inhibition of MAPK/ERK upregulated both full length TF (flTF) and soluble isoform TF (asTF) gene expression and cell-associated TF activity in breast cancer MDA-MB-231 cells. We explored the possible mechanisms, especially the possible interaction with EGFR and PI3K/Akt pathways.MethodsA plasmid containing TF promoter −2174 ~ +128 plus luciferase reporter gene was introduced into MDA-MB-231 cells to evaluate TF promoter activity. In order to study the interaction of these pathways, ERK inhibitor (PD98059), PI3K inhibitors (LY294002, wortmannin), Akt inhibitor (A6730), and EGFR inhibitor (erlotinib) as well as the corresponding siRNAs were used to treat MDA-MB-231 cells, and ovarian cancer OVCAR-3 and SKOV-3 cells. Quantitative PCR and western blot were used to determine TF expression. One stage clotting assays were used to measure pro-coagulation activity of the MDA-MB-231 cells.ResultsWe show that PI3K inhibitors LY294002, wortmannin and A6730 significantly inhibited TF promoter activity, and reduced TF mRNA and protein levels due to the inhibition of Akt phosphorylation. In contrast, ERK inhibitor PD98059 and ERK siRNA enhanced TF promoter activity by 2.5 fold and induced an increase in TF mRNA and protein levels in a dose dependent manner in these cells. The PI3K/Akt pathway was shown to be involved in PD98059-induced TF expression because the induction was inhibited by PI3K/Akt inhibitors. Most interestingly, the EGFR inhibitor erlotinib and EGFR siRNA also significantly suppressed PD98059- or ERK siRNA-induced TF promoter activity and TF protein expression. Similar results were found with ovarian cancer cells SKOV-3 and OVCAR-3. Furthermore, in MDA-MB-231, mRNA levels of asTF were regulated in a similar way to that of TF in response to the cell treatment.ConclusionsThis study showed a regulatory mechanism in which MAPK/ERK signals inhibit EGFR/PI3K/Akt-mediated TF expression in breast cancer MDA-MB-231 cells. The same regulation was observed in ovarian cancer OVCAR-3 and SKOV-3 cells. Interestingly, we observed that both flTF and asTF could be regulated in a parallel manner in MDA-MB-231. As the PI3K/Akt pathway and EGFR regulate TF expression in cancer cells, targeting these signaling components is expected to potentially inhibit TF expression-associated tumor progression.

Preeclampsia: the role of tissue factor and tissue factor pathway inhibitor

Preeclampsia (PE) is a multi-system disorder of human pregnancy, whose etiology remains poorly understood. Preeclamptic women are known to have an increased hypercoagulable state that result in excess fibrin deposition in several organs, which compromises their function. Tissue factor (TF) is the main physiological initiator of blood coagulation and its activity is regulated by a specific inhibitor known as Tissue factor pathway inhibitor (TFPI). Based on the important role of TF and TFPI in hemostasis, we hypothesize that their levels may change in the severe PE contributing to exacerbate hypercoagulable state. Some studies have assessed the balance between TF and TFPI in preeclamptic women, but results are inconsistent. Therefore, the aim of this study was to examine these inconsistencies and to assess TF and TFPI plasma levels in three groups of age matched women; pregnant with severe PE (n = 60), normotensive pregnant (n = 50) and normotensive non-pregnant women (n = 50). There was not significantly different among the three groups for TF plasma levels; severe PE women: 338.4 pg/mL (248.1-457.6), normotensive pregnant women: 301.5 pg/mL (216.4-442.9) and normotensive non-pregnant women 393 pg/mL (310.3-522.9). TFPI plasma levels were higher in severe PE comparing to normotensive pregnant women and normotensive non-pregnant women, 115.8 ng/mL (75-149.8); 80.3 ng/mL (59.6-99.7) and 74.5 ng/mL (47.1-98.0), respectively No difference was found between normotensive pregnant women and normotensive non-pregnant women. As for gestational age, a significant difference in TFPI levels was found between severe PE and normotensive pregnant women up to the 33rd week of pregnancy (p = 0.001), and severe PE and non-pregnant women up to the 34th (p = 0.01). In summary, our results indicated that TF plasma levels did not vary in the studied groups, while TFPI plasma levels were significantly increased in severe PE compared to normotensive pregnant and normotensive non-pregnant women. So, our data do not explain the exacerbated hypercoagulability state observed in severe PE. Further studies evaluating genes expression, TF activity and antigen, total and free TFPI and TFPI-2, both in plasma and obstetric tissues, throughout the pregnancy in PE (mild and severe forms) are required.

Splice variants of Tissue Factor and integrin-mediated signaling

Full-length Tissue Factor (flTF) – the obligatory co-factor for the serine protease (factor) VII/VIIa – serves as the initiator of blood coagulation. The flTF/VIIa complex triggers a sequence of proteolytic events that lead to the formation of a hemostatic plug. Aside from hemostatic maintenance, flTF can contribute to thrombogenesis in some settings. The proteolytic properties of the flTF/VIIa complex (as well as the flTF/VIIa/Xa complex) account for non-hemostatic functions of flTF, largely exerted through activation of intracellular signaling via Protease Activated Receptors (PARs). The flTF-PAR nexus impacts several kinases highly significant in the pathobiology of cancer and cardiovascular disease. Over the past decade, many advances have been made in the understanding of PAR-mediated functions of flTF, an important highlight of which was the finding that a sub-set of integrins – a diverse family of integral membrane proteins – cross-regulate flTF-elicited signaling. Concomitantly, an alternatively spliced TF form (asTF) was discovered in human and mouse. Initial studies characterizing asTF revealed that it is differentially expressed during development, continuously present in circulating blood and solid tissues, and possesses very low pro-coagulant activity. Hypomorphic nature of asTF's cofactor activity is the source of an ongoing controversy over whether asTF is pro-coagulant, and how it may contribute to hemostatic maintenance and/or its aberrations. Very recently, a novel concept emerged in asTF biology: asTF can evidently trigger intracellular signaling that promotes the formation of new vessels from the existing ones (angiogenesis) and monocyte-endothelial interactions, via interaction with integrins. We provide a brief overview of the fl/asTF-integrin nexus with an emphasis on asTF's non-proteolytic, integrin-mediated biological activity.

Decryption of tissue factor

Tissue factor (TF) is a transmembrane protein which, in complex with factor (F)VIIa, initiates blood coagulation. Numerous studies have determined TF epitopes and individual amino acids which play an important role in the TF/FVIIa complex formation and its activity towards natural substrates. However the subject of cell-surface TF activity remains controversial. It has been almost commonly accepted that TF on the cell surface has low (if any) activity, i.e. is encrypted and requires specific conditions/reagents to become active, i.e. decrypted. One of the leading theories suggests that cell membrane lipid composition plays a crucial role in TF decryption, whereas another assigns the key role to the formation of the Cys186-Cys209 disulfide bond. Despite a number of studies published from several laboratories, the role of this bond in the activity of the TF/FVIIa complex remains elusive and controversial. One of the causes of this controversy could be related to the lack of specificity of the reagents used for the cell treatment leading to possible alterations in other cell surface proteins and cell membrane environment. In conclusion, the influence of the Cys186-Cys209 this bond on cell surface TF function remains unclear.

Tissue factor and cardiomyocytes

Tissue factor, the initiator of blood coagulation, plays a major role in the protection of several organs against bleeding. Cardiomyocytes and fibroblasts constitutively express tissue factor. Besides its role for maintain cardiac hemostasis TF has been suggested to have several other important functions in the cardiac muscle. This review summarizes the different roles of TF in the heart with a special focus on pathological conditions.

Alternatively spliced tissue factor and full-length tissue factor protect cardiomyocytes against TNF-α-induced apoptosis

Tissue Factor (TF) is expressed in various cell types of the heart, such as cardiomyocytes. In addition to its role in the initiation of blood coagulation, the TF:FVIIa complex protects cells from apoptosis. There are two isoforms of Tissue Factor (TF): "full length" (fl)TF--an integral membrane protein, and alternatively spliced (as)TF--a protein that lacks a transmembrane domain and can thus be secreted in a soluble form. Whether asTF or flTF affects apoptosis of cardiomyocytes is unknown. In this study, we examined whether asTF or flTF protects murine cardiomyocytes from TNF-α-induced apoptosis. We used murine cardiomyocytic HL-1 cells and primary murine embryonic cardiomyocytes that overexpressed either murine asTF or murine flTF, and stimulated them with TNF-α to initiate cell death. Apoptosis was assessed by annexin-V assay, propidium iodide assay, as well as activation of caspase-3 and -9. In addition, signaling via integrins, Akt, NFκB and Erk1/2, and gene-expression of Bcl-2 family members were analyzed. We here report that overexpression of asTF reduced phosphatidylserine exposure upon TNF-α-stimulation. asTF overexpression led to an increased expression and phosphorylation of Akt, as well as up-regulation of the anti-apoptotic protein Bcl-x(L). The anti-apoptotic effects of asTF overexpression were mediated via α(V)β(3)/Akt/NFκB signaling and were dependent on Bcl-x(L) expression in HL-1 cells. The anti-apoptotic activity of asTF was also observed using primary cardiomyocytes. Analogous yet less pronounced anti-apoptotic sequelae were observed due to overexpression of flTF. Importantly, cardiomyocytes deficient in TF exhibited increased apoptosis compared to wild type cells. We propose that asTF and flTF protect cardiomyocytes against TNF-α-induced apoptosis via activation of specific signaling pathways, and up-regulation of anti-apoptotic members of the Bcl-2 protein family.