Interaction Of Tissue Factor Research Articles

Direct red blood cell effect on thrombosis is dependent on the interaction of tissue factor and calcium with membrane phosphatidylserine.

Prior literature has implicated red blood cells (RBCs) in the initiation of thrombosis and suggests that posttransfusion hypercoagulability may occur secondary to the effects of RBCs. Elevated serum tissue factor is a known sequelae of acute trauma. Phosphatidylserine (PS) is a prothrombotic phospholipid present within the RBC cell membrane. We hypothesized that RBC aggregation is dependent on the interaction between RBC membrane bound (exposed) PS, extracellular calcium, and tissue factor. Human whole blood (WB) was separated into components, including RBCs and platelet-rich plasma (PRP). Whole blood, PRP, and RBCs underwent impedance aggregometry utilizing arachidonic acid (AA), ADP, collagen, calcium, and tissue factor (TF)-based agonists. Red blood cells then underwent impedance aggregometry utilizing combined calcium and TF agonists. Red blood cells were pretreated with Annexin V, a known PS blocking agent, and underwent impedance aggregometry with combined calcium and TF agonists to determine if the mechanism of calcium/TF-induced RBC aggregability is dependent on PS. Red blood cells treated with calcium, TF, calcium+TF, and pre-treated with Annexin V followed by calcium+TF were perfused through an in vitro model of pulmonary microcirculatory flow. Red blood cell aggregation was significantly higher than that of WB and PRP when utilizing a TF agonist, an effect unique to TF. The combination of calcium and TF demonstrated significantly higher RBC aggregation than either agonist alone. Pretreatment with Annexin V resulted in a significantly reduced aggregability of RBC following treatment with TF + calcium. Red blood cells aged to 42 days did not exhibit significant change in aggregation. Exposure to calcium and TF significantly reduced time to thrombosis of RBCs perfused through a pulmonary microcirculatory model. Treatment with both TF and calcium synergistically induces RBC aggregation. Phosphatidylserine appears to play an integral role in the TF/calcium-based, age-independent RBC aggregation response. Red blood cells treated with TF + calcium exhibit more rapid thrombus formation in an in vitro model of pulmonary microcirculatory perfusion.

Regulation of coagulation activation in newly diagnosed AML by the heme enzyme myeloperoxidase

IntroductionPatients with acute myeloid leukemia (AML) are at increased risk of thrombohemorrhagic complications. Overexpressed tissue factor (TF) on AML blasts contributes to systemic coagulation activation. We have recently shown that the heme enzyme myeloperoxidase (MPO) negatively regulates TF procoagulant activity (PCA) on myelomonocytic cells in vitro. We now aimed to further characterize the functional interaction of MPO and TF in AML in vivo. MethodsWe prospectively recruited 66 patients with newly diagnosed AML. TF PCA of isolated peripheral blood mononuclear cells (PBMC) was assessed by single-stage clotting assay in the presence or absence of inhibitors against MPO catalytic activity (ABAH) or against MPO-binding integrins (anti-CD18). MPO in plasma and in AML blasts was measured by ELISA, and plasma D-dimers and prothrombin fragment F1+2 were quantified by automated immunoturbidimetric and chemiluminescence assays, respectively. ResultsPatients with AML had significantly higher MPO plasma levels compared to healthy controls and exhibited increased levels of D-dimers and F1+2. In vivo thrombin generation was mediated by TF PCA on circulating PBMC. Ex vivo incubation of isolated PBMC with ABAH or anti-CD18 antibody resulted in either increased or decreased TF PCA. The strong and robust correlation of F1+2 with TF PCA of circulating PBMC was abrogated at MPO plasma levels higher than 150 ng/mL, indicating a modulatory role for MPO on TF-mediated in vivo thrombin generation above this threshold. ConclusionOur study indicates that catalytically active MPO released by circulating myeloblasts regulates TF-dependent coagulation in patients with newly diagnosed AML in a CD18-dependent manner.

Hemophilia: The Past, the Present, and the Future.

Hemophilia: The Past, the Present, and the Future.

Tissue Factor Expression As a Prognostic Marker in Patients with B-Cell Malignant Lymphoma

Background. Studies in tumor cell lines have shown an increased expression of TF. Expression of TF in adenocarcinoma of the pancreas is associated with tumor progression. It has been described that between 5% and 10% of patients with malignant lymphoma (ML) may develop VTD. The mechanisms of ML-associated thrombosis are unknown, however, expression of functional TF may increase as much as 75-fold in peripheral blood monocytes, particularly during relapse. As expected, thrombosis was found in patients with the highest TF levels. The role of TF in the biology of ML has not yet been determined. Considering indirect evidence, it seems that, besides its procoagulant effects, TF may have a role in tumor progression.Objective. The purpose of the present study was to elucidate whether the interaction of tissue factor and TF/VIIa could induce relapsed in non Hodgkin Lymphoma patients.Patients and Methods. We therefore performed a prospective study (cohort) to evaluate the risk of TF in non Hodgkin Lymphoma patients without a previous episode of venous thromboembolism. 59 patients 30 women and 29 men were enrolled in this study. The median age was 43.3 ± 14.5 (range 20 to 59). The TF and FT/VIIa activity was performed on fresh plasma (American Diagnostics, USA), and other tests (PC, PS, AT and activated protein C resistance) (STAGO, Asnieres, Francia).Results.59 patients with B-cell ML, 30 women and 29 men with a mean age of 43.3 years (ranges 20 to 59). According to the Ann Arbor Staging system, 44% percent of patients (n=26) had a stage IV and only 14% at stage I. ML was mostly limited to lymph nodes (74%).Results of hemostatic markers were between the normal ranges. However, concentrations of factors involved in the initiation phase of fluid phase of blood coagulation (TF, FT/VIIa complex, and FVII) had an abnormal distribution. None case with APCR was found.Comparison between complete remission and relapsing patients. Thirty-one patients (52.5%) achieved complete remission at the end of follow-up and 28 (47.5%) relapsed. Mean follow up for patients with complete remission was 48 months vs. 18 months for relapsing patients (p <0.001). After comparing TF concentrations between the groups, relapsing patients had higher levels of TF (p=0.005). TF/FVIIa complex levels were also significantly higher in patients with relapsing disease (p=0.019). As expected LDH levels were significantly lower in patients with complete remission (p=0.0001).TF levels and response to treatment. Twenty-nine patients had normal TF levels. Relapses were more frequently found in patients with elevated TF concentrations (n=19) as compared with patients with normal TF levels (n=9) (p=0.013). Elevated TF/FVIIa complexes levels were also more frequently seen among patients with relapsing ML (n=32) as compared with patients with complete remission (n=27) (p=0.046).As expected, other factors associated with a poor prognosis were DHL levels (p=0.001) and IPI (p=0.001).Discussion. TF and TF/FVIIIa concentrations were significantly different in patients who achieved complete remission as compared with those with relapse after treatment. As expected for the staging of patients included LDH serum levels as well as IPI score were also significantly different between these two groups of patients. Moreover, estimation of the relative risks associated with relapsing disease showed that also these four variables were associated with relapsing disease. However, linear regression analysis showed that TF, LDH, and IPI but not TF/FVIIa complexes remained as a risk factor for relapse in patients with ML. To our knowledge, we have demonstrated for the first time that high TF concentration is a prognosis factor for relapse in patients with B-cell ML.Our results seem consistent with previous reports regarding an association between the increased TF expression and advanced staging of the malignant disease. It has been demonstrated the presence of high plasma levels of TF and FVIII in patients with solid tumors suggesting that it may be an important mechanism to activate blood coagulation system in cancer patients. Tumor cells may also induce TF expression on the cell surface of peripheral blood monocytes and macrophages. DisclosuresNo relevant conflicts of interest to declare.

The effects of citrate dialysate in hemodialysis on polymorphonuclear elastase interaction with tissue factor and its inhibitor.

This study aimed to investigate whether hemodialysis (HD) affects tissue factor (TF), tissue factor pathway inhibitor (TFPI), and polymorphonuclear elastase (PMNE) in endstage renal disease (ESRD) patients when eliminating the effects of heparin. Also, to explore the interaction of TF, TFPI, and PMNE throughout a single HD session. We enrolled 57 ESRD patients who had undergone hemodialysis for >3 months as an experimental group. Plasma levels of TF, TFPI and PMNE were measured by ELISA in 24 ERSD patients on intermittent HD using low-molecular-weight heparin (LMWH) as anticoagulation (LMWH group) and 33 ESRD patients using citrate as anticoagulation (citrate group) at the start and at 1, 2 and 5 h of the HD session. Meanwhile,28 ESRD patients not on dialysis were enrolled as a control group and fasting venous blood samples were taken in the morning. Compared with the control group, the plasma TFPI levels of the LMWH group and the citrate group were significantly higher (P=0.000, P=0.002, respectively) under baseline conditions as well as the plasma PMNE levels (P=0.001, P=0.02, respectively), whereas TF showed no difference (P=0.186). During HD with citrate, plasma TFPI decreased slightly (P=0.012) and PMNE increased significantly (P=0.008) at 1 h. The plasma TFPI levels of the citrate group correlate with PMNE at 2 and 5 h (P=0.001, P=0.008, respectively). ESRD patients on HD have significantly higher TFPI and PMNE levels compared to patients not on HD under baseline conditions, while TF levels were similar between the three groups. TFPI and PMNE are differently regulated, but the plasma levels correlated during HD in the citrate group. It might be possible that PMNE plays a role in anticoagulative activity through TFPI.

Crystal structure of tissue factor in complex with antibody 10H10 reveals the signaling epitope

Tissue factor (TF) initiates the extrinsic pathway of blood coagulation through sequential binding and activation of coagulation factors VII (FVII) and X (FX). In addition, through activation of G-protein-coupled protease activated receptors (PARs) TF induces cell signaling that is related to cancer, angiogenesis and inflammation. Monoclonal antibodies (mAbs) proved to be a useful tool for studying the interplay between TF signaling and coagulation. MAb 10H10 is unique in that it blocks the signaling pathway and thus inhibits angiogenesis and tumor growth without interfering with coagulation. It was also presumed that mAb 10H10 recognizes the cryptic pool of TF devoid of procoagulant activity. The crystal structure of the 10H10 Fab was determined in the absence and in the presence of the TF extracellular domain (ECD). The structures show that the antibody operates by the key-and-lock mechanism causing no conformational changes in either Fab or TF. The TF:10H10 interface is extensive and includes five segments of TF in both the N-terminal and C-terminal domains of the ECD. Neither the known epitope of FVII, nor the putative epitope of FX overlaps with the 10H10 binding site. The 10H10 epitope points to the likely location of the PAR2 exosite. It is also the hypothetical site of TF interaction with integrins that may play a major role in the encryption-decryption process.

Investigation of the Filamin A-Dependent Mechanisms of Tissue Factor Incorporation into Microvesicles.

We have previously shown that phosphorylation of tissue factor (TF) at Ser253 increases the incorporation of TF into microvesicles (MVs) following protease-activated receptor 2 (PAR2) activation through a process involving filamin A, whereas phosphorylation of TF at Ser258 suppresses this process. Here, we examined the contribution of the individual phosphorylation of these serine residues to the interaction between filamin A and TF, and further examined how filamin A regulates the incorporation of TF into MVs. In vitro binding assays using recombinant filamin A C-terminal repeats 22-24 with biotinylated phospho-TF cytoplasmic domain peptides as bait showed that filamin A had the highest binding affinities for phospho-Ser253 and double-phosphorylated TF peptides, while the phospho-Ser258 TF peptide had the lowest affinity. Analysis of MDA-MB-231 cells using an in situ proximity ligation assay revealed increased proximity between the C-terminus of filamin A and TF following PAR2 activation, which was concurrent with Ser253 phosphorylation and TF-positive MV release from these cells. Knock-down of filamin A expression suppressed PAR2-mediated increases in cell surface TF procoagulant activity without reducing cell surface TF antigen expression. Disrupting lipid rafts by pre-incubation with methyl-β-cyclodextrin prior to PAR2 activation reduced TF-positive MV release and cell surface TF procoagulant activity to the same extent as filamin A knock-down. In conclusion, this study shows that the interaction between TF and filamin A is dependent on the differential phosphorylation of Ser253 and Ser258. Furthermore, the interaction of TF with filamin A may translocate cell surface TF to cholesterol-rich lipid rafts, increasing cell surface TF activity as well as TF incorporation and release into MVs.

Proangiogenic TF-FVIIa-PAR2 Signaling Requires Matriptase-Independent Integrin Interaction

The close link between coagulation activation and cancer progression is supported by clinical and experimental studies. A central molecular pathways by which tumor cells interact with the hemostatic system is through the expression of the cell surface receptor tissue factor (TF) that in complex with coagulation factor VIIa (FVIIa) triggers the extrinsic pathway of blood coagulation, contributes to cancer associated thrombosis, and promotes direct tumor cell signaling through protease-activated receptors (PARs). Genetic and pharmacological evidence shows that epithelial and tumor cell TF-FVIIa signaling induces a diverse set of proangiogenic and immune modulatory cytokines, chemokines and growth factors. However, the mechanistic details of this signaling pathway remain incompletely understood. Furthermore, a recent study has suggested that the TF-FVIIa complex does not directly cleave PAR2, but rather induces PAR signaling through initiating a cell surface protease cascade involving the transmembrane serine protease matriptase. Here, we employ mutants of FVIIa to characterize the roles of cell surface proteolysis and endosomal signaling by TF in keratinocytes and cancer cells.We studied TF-expressing MDA-MB-231 breast cancer cells, A7 melanoma cells transduced with TF and PAR2, and non-tumorigenic HaCaT keratinocytes. Stimulation with 10 nM FVIIa or 5 nM recombinant matriptase promoted productive PAR2 signaling in all three cell types as assessed by quantification of cytokine IL-8 mRNA up-regulation. Addition of the serine protease inhibitor aprotinin abolished IL-8 upregulation by recombinant matriptase, but not by FVIIa. Importantly, matriptase was expressed by HaCaT and A7 melanoma cells, whereas MDA-MB-231 cells did not express matriptase, while responding to FVIIa with efficient proangiogenic signaling. HaCaT and A7 cells showed shedding of the matriptase protease domain following incubation with FVIIa, suggesting activation of the serine protease. However, the signaling-defective FVIIa Q40A mutant also promoted matriptase shedding without inducing IL-8. We had previously shown that FVIIa induces association of TF with integrin β1 hetorodimers. We characterized an integrin-binding deficient FVIIa mutant, E26A, rendered incapable to interact with active integrin β1, resulting in defective TF-FVIIa internalization, TF-FVIIa stimulated cell migration and induction of IL-8. FVIIa E26A promoted matriptase shedding as observed with wild-type FVIIa. We found that inhibition of the small rho GTPase arf6, which controls cellular trafficking of β1 integrins, by pharmacological inhibitors or expression of a dominant negative arf6 mutant prevented IL-8 induction by TF-FVIIa, but not by direct stimulation of PAR2 with agonist peptide. Pharmacological activation of arf6 or expression of arf6 constitutive active mutant on the other hand promoted translocation of TF-FVIIa, which unlike PAR2 agonist peptide induced delayed and prolonged MAPK activation downstream of PAR2. This data show that TF-FVIIa triggers PAR2 signaling dependent on integrin-arf6-regulated trafficking and prolonged activation of endosomal signaling.Our data support the conclusion that the FVIIa induced interaction of TF with integrins and endocytosis of the TF-FVIIa-integrin complex plays a pivotal role in inducing proangiogenic cytokines downstream of proteolytic cleavage of PAR2. Mutants of FVIIa that are defective in supporting this pathway fully promote cell surface matriptase activation, but fail to upregulate tumor cell proangiogenic responses or tumor cell migration. These results argue against a role for matriptase as an intermediary in TF-FVIIa induced PAR2 activation, but do not exclude that matriptase modulates homeostatic epithelial responses through PAR2 signaling in non-malignant cells. These data furthermore suggest that matriptase inhibitors cannot substitute for targeting TF as potential therapeutic strategy to attenuate cancer proangiogenic signaling of the hemostatic system DisclosuresOstergaard:Novo Nordisk A/S: Employment. Ruf:Iconic Therapeutics: Consultancy.

EGFP–EGF1-conjugated nanoparticles for targeting both neovascular and glioma cells in therapy of brain glioma

As neovascular and glioma cells were closely associated and might be mutually promoted in glioma growth, a dual-targeting strategy targeting to both neovascular and glioma cells would be more promising as compared with those targeting one of them. In this study, we reported a drug delivery system where nanoparticles were decorated with EGFP–EGF1 (ENP), a fusion protein derived from factor VII with special affinity for tissue factor (TF) over-expressed in glioma tissues, to facilitate anti-glioma delivery of paclitaxel (PTX) by targeting both neovascular and glioma cells. In vitro protein binding assay demonstrated that EGFP–EGF1 bound well to C6 cells and perturbed human umbilical vein endothelial cells (HUVEC) with a concentration-dependent manner but not to unperturbed HUVEC. EGFP–EGF1–TF interaction significantly enhanced nanoparticles uptake by perturbed HUVEC and glioma C6 cells as well as nanoparticles penetration in C6 glioma spheroids, and thus improved the cytotoxicity of their payload in both monolayer cells and glioma spheroids models. In vivo imaging of glioma-bearing mice demonstrated the specific accumulation of ENP in glioma tissues. In vivo distribution of nanoparticles intuitively showed ENP mainly sited in both extravascular glioma cells and neovascular cells. Pharmacodynamic results revealed that PTX-loaded ENP (ENP–PTX) significantly prolonged the median survival time of glioma-bearing mice compared with that of any other group. TUNEL assay and H&E staining showed that ENP–PTX treatment induced significantly more cell apoptosis and tumor necrosis compared with other treatments. In conclusion, the results of this contribution demonstrated the great potential of EGFP–EGF1-functionalized nanoparticles for dual-targeting therapy of brain glioma.

Posttranslational modifications of tissue factor

Tissue factor (TF), a membrane protein, is an initiator of blood coagulation in vivo. In this review we discuss how posttranslational modifications affect activity and other properties of TF. Glycosylation of the extracellular domain and the composition of carbohydrates at three glycosylation sites have an influence on TF activity in the extrinsic FXase by increasing the rate of FX proteolysis. No influence of TF glycosylation on the activity of the FVIIa/TF complex towards small synthetic substrates was observed, suggesting that glycosylation has no effect on TF interaction with FVIIa. There are no published data suggesting a direct influence of phosphorylation or palmitoylation in the cytoplasmic domain on TF procoagulant activity. There has been a debate in the recent literature related to the role and formation of the Cys¹⁸⁶-Cys²⁰⁹ disulfide bond. Published opinions from various laboratories range from this bond being essential for the expression of cell TF activity to having no role in it. Overall, it is clear that some modifications of TF have an effect on TF procoagulant activity, signaling functions and trafficking. The influences of other modifications are debatable.

Induction of Endothelial Cell Proliferation by Recombinant and Microparticle-Tissue Factor Involves β1-Integrin and Extracellular Signal Regulated Kinase Activation

Increased levels of circulating tissue factor (TF) in the form of microparticles increase the risk of thrombosis. However, any direct influence of microparticle-associated TF on vascular endothelial cell proliferation is not known. In this study, the influence of recombinant and microparticle-associated TF on endothelial cell proliferation and mitogen-activated protein kinase signaling mechanisms was examined. Incubation of human coronary artery endothelial cells with lipidated recombinant full-length TF, or TF-containing microparticles (50 to 200 pmol/L TF), increased the rate of cell proliferation and induced phosphorylation of extracellular signal regulated kinase 1 in a TF-dependent manner. Inhibition of extracellular signal regulated kinase 1/2 using PD98059 or extracellular signal regulated kinase 1/2 antisense oligonucleotides or inhibition of c-Jun N-terminal kinase reduced recombinant TF-mediated cell proliferation. PD98059 also reduced cell proliferation in response to TF-containing microparticles. Inclusion of FVIIa (5 nmol/L) and FXa (10 nmol/L) or preincubation of cells with an inhibitory anti-FVIIa antibody had no additional influence on TF-mediated cell proliferation. However, preincubation of exogenous TF with a beta1-integrin peptide (amino acids 579 to 799) reduced TF-mediated proliferation. High concentrations of recombinant or microparticle-associated TF stimulate endothelial cell proliferation through activation of the extracellular signal regulated kinase 1/2 pathway, mediated through a novel mechanism requiring the interaction of exogenous TF with cell surface beta1-integrin and independent of FVIIa.

Thrombophilia and placenta-mediated pregnancy complications: from the bench to bedside to policy

While it is well known that thrombin plays a central role in the coagulation component of hemostasis [1] a developing story is emerging that demonstrates thrombin’s central role in placental development. Tissue Factor (TF), the most important initiator of coagulation, is constitutively expressed on almost all cells other than endothelial cells [2]. Endothelial cells line the inner layer of blood vessels and provide a physical barrier between TF laden sub-endothelial tissues and coagulation ready blood. When the endothelial barrier is disrupted (e.g. vascular injury) coagulation ensues. FVIIa circulates in minute quantities in normal plasma at the ready to encounter TF. Coagulation activation is initiated through interaction of Tissue Factor, calcium and circulating FVIIa which then convert factor X to Xa and factor IX to IXa. Xa then complexes with Va and calcium on a phospholipids surface to form the Tenase complex. The Tenase complex then converts prothrombin to thrombin. Next, coagulation activation is maintained by thrombin activating factors XI, V and VIII to generate a positive feedback cycle that leads to a thrombin burst which then cleaves sufficient fibrinogen to form adequate fibrin for a hemostatic plug. Thrombin also activates platelets via binding to the PAR-4 receptor on platelets. Without an adequate anticoagulant mechanism unchecked thrombin generation would lead to disseminated coagulation throughout the vascular system. The

Inhibition of tissue factor signaling suppresses tumor growth

Coagulation activation by tissue factor (TF) is implicated in cancer progression, cancer-associated thrombosis and metastasis. The role of direct TF signaling pathways in cancer, however, remains incompletely understood. Here we address how TF contributes to primary tumor growth by using a unique pair of isotype-matched antibodies that inhibit either coagulation (monoclonal antibody [Mab]-5G9) or direct signaling (Mab-10H10). We demonstrate that the inhibitory antibody of direct TF-VIIa signaling not only blocks TF-VIIa mediated activation of PAR2, but also disrupts the interaction of TF with integrins. In epithelial and TF-expressing endothelial cells, association of TF with beta1 integrins is regulated by TF extracellular ligand binding and independent of PAR2 signaling or proteolytic activity of VIIa. In contrast, alpha3beta1 integrin association of TF is constitutive in breast cancer cells and blocked by Mab-10H10 but not by Mab-5G9. Mab-5G9 has antitumor activity in vivo, but we show here that Mab-10H10 is at least as effective in suppressing human xenograft tumors in 2 different models. Breast tumor growth was also attenuated by blocking PAR2 signaling. These results show that tumor cell TF-PAR2 signaling is crucial for tumor growth and suggest that anti-TF strategies can be applied in cancer therapy with minor impairment of TF-dependent hemostatic pathways.

Tissue factor/FVII regulates doxorubicin-induced apoptosis in glioblastoma via activating PI3K/Akt signaling

To investigate the role of tissue factor (TF) in chemotherapeutic reagent — induced apoptosis on human glioblastoma and explore its mechanism. The expression of TF was examined by Western blotting. The cytotoxicity of doxorubicin was determined by WST assay. The activation of Caspase-3 and PARP induced by adoxorubicin were tested by Western blotting. Human glioblastoma cell line U373MG expressed high level of TF while LN-229 was with low-TF level. The chemotherapeutic reagent doxorubicin revealed stronger cytotoxic effect on high-TF U373MG cells than low-TF LN-229 cells. Enforced strong expression of TF was achieved by transfection of TF-pcDNA3 combinant on LN-229 cells in a dose-dependent manner. Enforced TF expression in transfected LN-229 cells not only impaired the doxorubicin-induced cleavage of Caspase-3 and PARP, but also inhibited the cytotoxic effect of doxorubicin. Furthermore, activation of Akt was strong in high-TF U373MG cells but weak in low-TF LN-229 cells. Incubation of factor VII (FVII) with enforced TF-expressing LN-229 cells increased the phosphorylation of Akt in a time-dependent manner. These results suggest that over-expression of TF on glioblastoma could inhibit doxorubicin-induced apoptosis. Interaction of FVII and TF activates the downstream PI3K/Akt pathway. Tumor-derived over-expression of TF might play a role in chemotherapy resistance in glioblastoma, at lest in part, by activating PI3K/Akt-mediated survival and anti-apoptotic mechanism through the interaction of TF/FVII signaling.

Tissue Factor Coagulant Function Is Enhanced by Protein-disulfide Isomerase Independent of Oxidoreductase Activity

Protein-disulfide isomerase (PDI) switches tissue factor (TF) from coagulation to signaling by targeting the allosteric Cys186-Cys209 disulfide. Here, we further characterize the interaction of purified PDI with TF. We find that PDI enhances factor VIIa-dependent substrate factor X activation 5-10-fold in the presence of wild-type, oxidized soluble TF but not TF mutants that contain an unpaired Cys186 or Cys209. PDI-accelerated factor Xa generation was blocked by bacitracin but not influenced by inhibition of vicinal thiols, reduction of PDI, changes in redox gradients, or covalent thiol modification of reduced PDI by N-ethylmaleimide or methyl-methanethiosulfonate, which abolished PDI oxidoreductase but not chaperone activity. PDI had no effect on fully active TF on either negatively charged phospholipids or in activating detergent, indicating that PDI selectively acts upon cryptic TF to facilitate ternary complex formation and macromolecular substrate turnover. PDI activation was reduced upon mutation of TF residues in proximity to the macromolecular substrate binding site, consistent with a primary interaction of PDI with TF. PDI enhanced TF coagulant activity on microvesicles shed from cells, suggesting that PDI plays a role as an activating chaperone for circulating cryptic TF.

Disulfide Switch Alters Receptor Signals

Coagulation of the blood is regulated by a member of the cytokine receptor family known as tissue factor (TF). Tissue factor associates with the protease factor VIIa and leads to activation of a proteolytic cascade that results in cleavage of fibrinogen and blood coagulation. But the TF-VIIa complex also initiates signaling through the protease-activated receptor 2 (PAR2), which influences a range of biological processes, including inflammation, angiogenesis, and vascular function. Ahamed et al . now report that a disulfide bond between two cysteine residues on the extracellular portion of TF acts as a switch to promote one or the other of these two actions of TF--coagulation or cell signaling through PAR2. The authors used a set of antibodies that recognized distinct epitopes on TF and dose-response experiments to show that two distinct pools of TF regulated coagulation (measured as proteolytic generation of factor Xa) or PAR2 signaling [measured by monitoring phosphorylation of ERKs (extracellular signal–regulated kinases)]. Studies with mutant TF supported the authors’ suspicion that a conformational change in TF might result from formation or breakage of the Cys 186 -Cys 209 disulfide bond. Furthermore, TF was found to associate with the enzyme protein disulfide isomerase (PDI). Reduction of the disulfide bond by PDI appeared to suppress coagulation, but TF retained its signaling activity through PAR2. Conversely, an antibody that inhibited interaction of TF with PAR2 inhibited signaling but allowed TF to function in control of coagulation. Pharmacological inhibition of PDI inhibited signaling through PAR2 initiated by the TF-VIIa complex, but not activation of the coagulation cascade. The findings may have therapeutic implications in that they demonstrate that it is possible to inhibit signaling initiated by TF-VIIa without compromising regulation of coagulation. J. Ahamed, H. H. Versteeg, M. Kerver, V. M. Chen, B. M. Mueller, P. J. Hogg, W. Ruf, Disulfide isomerization switches tissue factor from coagulation to cell signaling. Proc. Natl. Acad. Sci. U.S.A. 103 , 13932-13937 (2006). [Abstract] [Full Text]

The pathogenesis of venous thromboembolism in cancer: emerging links with tumour biology

Venous thromboembolism (VTE) is a frequent complication in individuals with cancer and is considered to be a cause of substantial mortality. Epidemiological studies identify malignancy as an independent VTE risk factor and show that cancer patients are at increased risk of both initial and recurrent VTE events. The risk due to cancer is compounded by the effects of chemotherapy and other treatments. The pathogenesis of cancer-associated VTE is complex involving multiple interactions between tumours and various components of haemostasis. The development of a systemic hypercoagulable state is considered a key pathogenetic feature and is attributed to tumour expression of tissue factor and other procoagulants, activation of vascular cells by tumour-derived cytokines and adhesive interactions between tumour cells and host cells. An increasing body of evidence indicates that the activation of haemostasis in malignant disease contributes to tumour growth and progression by stimulation of intracellular signalling pathways. The interaction of tissue factor, thrombin and other coagulation factors with protease activated receptor (PAR) proteins expressed by tumour cells and host vascular cells leads to the induction of genes related to the processes of angiogenesis, cell survival and cell adhesion and migration.

A potent anti-coagulant RNA aptamer inhibits blood coagulation by specifically blocking the extrinsic clotting pathway

A potent antidote-controlled aptamer, as an anticoagulant, has reportedly overcome the complications of acute bleeding by the administration of available anticoagulants. In the present study, we provide evidence that the aptamer binds specifically to factors IX and IXa and inhibits their functions. Furthermore, we demonstrated that the aptamer inhibits blood coagulation by interfering with the extrinsic pathway, blocking the complex of factor VIIa and tissue factor interactions with factor IX. The results from the previous and present studies suggest that the aptamer probably binds in the vicinity of the EGF1 and EGF2 domains of factor IX.

Estrogenic regulation of tissue factor and tissue factor pathway inhibitor in platelets

Oral estrogen treatment increases thrombotic risk. Tissue factor (TF), tissue factor pathway inhibitor (TFPI), and platelet interaction with leukocytes are important determinants of thrombogenesis. Therefore, the present study was designed to define and compare platelet TF and TFPI mRNA and adhesion protein expression in platelets derived from animals treated with different types of oral estrogens. Ovariectomized pigs were treated with 17beta-estradiol (2 mg/day), conjugated equine estrogen (CEE; 0.625 mg/day), or raloxifene (60 mg/day) for 4 wk. Compared with intact animals, ovariectomy and treatment differentially affected populations of leukocytes: neutrophils decreased whereas lymphocytes increased significantly 4 wk after ovariectomy and with 17beta-estradiol and CEE treatments; eosinophils increased only with 17beta-estradiol treatment. Content of TF protein increased in platelets from 17beta-estradiol- and raloxifene-treated pigs, whereas TF mRNA was detected only in platelets from 17beta-estradiol- and CEE treated pigs. TFPI mRNA increased in platelets after ovariectomy and estrogen treatment. Only a trace of TFPI protein was detected, but a higher-molecular-mass protein was observed in all treatment groups. Expression of CD40 and CD40 ligand increased with ovariectomy and decreased with 17beta-estradiol and CEE treatments more than with raloxifene. The ratio of activated to basal P-selectin expression decreased with ovariectomy and increased with raloxifene treatments. These results suggest that estrogenic formulations may affect individual thrombotic risk by different mechanisms that regulate TF and platelet-leukocytic interactions. These studies provide the rationale for evaluation of interactions among platelets and TF and TFPI expression on thrombin generation during estrogen treatment in humans.

Tissue Factor–Factor VIIa Signaling

How does tissue factor (TF), whose principle role is to support clotting factor VIIa (FVIIa) in triggering the coagulation cascade, affect various pathophysiological processes? One of the answers is that TF interaction with FVIIa not only initiates clotting but also induces cell signaling via activation of G-protein-coupled protease activated receptors (PARs). Recent studies using various cell model systems and limited in vivo systems are beginning to define how TF-VIIa-induced signaling regulates cellular behavior. Signaling pathways initiated by both TF-VIIa protease activation of PARs and phosphorylation of the TF-cytoplasmic domain appear to regulate cellular functions. In the present article, we review the emerging data on the mechanism of TF-mediated cell signaling and how it regulates various cellular responses, with particular focus on TF-VIIa protease-dependent signaling.