Beyond thrombosis: the impact of tissue factor signaling in cancer.

Tissue factor (TF) is the primary initiator of the coagulation cascade, though its effects extend well beyond hemostasis. When TF binds to Factor VII, the resulting TF:FVIIa complex can proteolytically cleave transmembrane G protein-coupled protease-activated receptors (PARs). In addition to activating PARs, TF:FVIIa complex can also activate receptor tyrosine kinases (RTKs) and integrins. These signaling pathways are utilized by tumors to increase cell proliferation, angiogenesis, metastasis, and cancer stem-like cell maintenance. Herein, we review in detail the regulation of TF expression, mechanisms of TF signaling, their pathological consequences, and how it is being targeted in experimental cancer therapeutics.

The effect of factor VIII deficiencies and replacement and bypass therapies on thrombus formation under venous flow conditions in microfluidic and computational models.

Clinical evidence suggests that individuals with factor VIII (FVIII) deficiency (hemophilia A) are protected against venous thrombosis, but treatment with recombinant proteins can increase their risk for thrombosis. In this study we examined the dynamics of thrombus formation in individuals with hemophilia A and their response to replacement and bypass therapies under venous flow conditions. Fibrin and platelet accumulation were measured in microfluidic flow assays on a TF-rich surface at a shear rate of 100 s⁻¹. Thrombin generation was calculated with a computational spatial-temporal model of thrombus formation. Mild FVIII deficiencies (5-30% normal levels) could support fibrin fiber formation, while severe (<1%) and moderate (1-5%) deficiencies could not. Based on these experimental observations, computational calculations estimate an average thrombin concentration of ∼10 nM is necessary to support fibrin formation under flow. There was no difference in fibrin formation between severe and moderate deficiencies, but platelet aggregate size was significantly larger for moderate deficiencies. Computational calculations estimate that the local thrombin concentration in moderate deficiencies is high enough to induce platelet activation (>1 nM), but too low to support fibrin formation (<10 nM). In the absence of platelets, fibrin formation was not supported even at normal FVIII levels, suggesting platelet adhesion is necessary for fibrin formation. Individuals treated by replacement therapy, recombinant FVIII, showed normalized fibrin formation. Individuals treated with bypass therapy, recombinant FVIIa, had a reduced lag time in fibrin formation, as well as elevated fibrin accumulation compared to healthy controls. Treatment of rFVIIa, but not rFVIII, resulted in significant changes in fibrin dynamics that could lead to a prothrombotic state.

TF/FVIIa/PAR2 promotes cell proliferation and migration via PKCα and ERK-dependent c-Jun/AP-1 pathway in colon cancer cell line SW620.

Our previous study has demonstrated that tissue factor-factor VIIa (TF/FVIIa) complex promotes the proliferation and migration of colon cancer cell line SW620 through the activation of protease-activated receptor 2 (PAR2). In the current study, the underlying molecular mechanisms of TF/FVIIa/PAR2 signaling in SW620 cells were further explored, with the focus on the role of activator protein-1 (AP-1) subunit c-Jun. The results revealed that PAR2-AP and FVIIa could upregulate c-Jun expression and c-Jun phosphorylation in SW620 cells in a time-dependent manner. The effect of FVIIa was significantly blocked by anti-TF and anti-PAR2 antibodies. Protein kinase Cα (PKCα) inhibitor safingol and extracellular signal-regulated kinase 1 and 2 (ERK1/2) inhibitor U0126 abrogated the activation of c-Jun. In contrast, Ca(2+) chelators EGTA and thapsigargin, and p38MAPK inhibitor SB203580 had no effect. Suppression of c-Jun/AP-1 activation using a natural inhibitor curcumin decreased the expression of caspase-3, MMP-9, and TF, as well as the proliferation and migration of SW620 cells induced by PAR2-AP or FVIIa. Collectively, our findings suggest that c-Jun/AP-1 activation is required for TF/FVIIa/PAR2-induced SW620 cell proliferation and migration. PKCα and ERK1/2 are located upstream of c-Jun/AP-1 in this signaling pathway. Pharmacological inhibition of this pathway might be a novel strategy for colon cancer therapy.

Active site-inactivated factor VIIa inhibits nuclear factor kappa B activation in intestinal ischemia and reperfusion.

BACKGROUND: Intestinal ischemia and reperfusion (I/R) injury is a pivotal mechanism in critical illness and in the development of multiple organ dysfunction syndrome, in which the nuclear factor kappa B (NF-κB) activation plays a central role. Intestinal I/R injury initiates the extrinsic tissue factor or factor VIIa-dependent pathway of coagulation, also of importance in multiple organ dysfunction syndrome. Our aim was to analyze NF-κB activation in I/R injury in the rat intestine and in two main "shock" organs, that is, the liver and lungs. Pretreatment with active site-inactivated factor VII (FVIIai), an inhibitor of the extrinsic pathway, was evaluated. MATERIALS AND METHODS: NF-κB activation was analyzed using enzyme-linked immunosorbent assay (ELISA) and electrophoretic mobility shift assay (EMSA) studies of nuclear extracts from the intestine, liver, and lungs in rats subjected to intestinal I/R injury. FVIIai was given 90 min before the induction of intestinal ischemia. RESULTS: I/R induced NF-κB p65 activation in all three organs, especially in the liver. Pretreatment with FVIIai counteracted NF-κB activation in all three tissues studied. A commercially available ELISA for (human) NF-κB p65 and EMSA gave parallel results. CONCLUSIONS: I/R injury in the rat intestine induces a pronounced activation of NF-κB p50 or p65 in the small intestine and in the liver and lungs. The NF-κB activation is especially pronounced in the liver and plays a central role in the regulation of transcription of cytokines, adhesion molecules, and chemokines. ELISA for (human) NF-κB p65 and "gold standard" EMSA gave parallel results. Pretreatment with FVIIai completely counteracted NF-κB activation in the intestine and liver, although not in the lungs.

Tissue factor and factor VIIa--hemostasis and beyond.

Initiation of the coagulation cascade via exposure of active tissue factor (TF) to blood and formation of the factor VIIa/TF complex is essential for hemostasis and is an initial procoagulant signal in thrombosis. As of early 2012, over 20,000 articles listed on PubMed describe advances in the understanding of TF biology in the settings of hemostasis and thrombosis, as well as in signaling events in cancer, sickle cell anemia, hyperlipidemia, and a broad spectrum of inflammatory disorders. It is both inspiring and humbling, then, to consider not only what has been learned about TF regulation, but also the number of questions still remaining about its role in physiology. This supplement reviews both well-accepted and currently-controversial topics in coagulation and factor VIIa/TF biology, with particular foci on non-hemostatic roles of TF, innovative approaches for the treatment of hemophilia, and novel in vivo models of bleeding and thrombosis.

Tissue factor/factor VIIa pathway mediates coagulation activation in induced-heat stroke in the baboon.

OBJECTIVE: Excessive activation of coagulation, which can culminate in overt disseminated intravascular coagulation, is a prominent feature of heat stroke. However, neither the mechanism that initiates the coagulation activation nor its pathogenic role is known. We examined whether the tissue factor/factor VIIa complex initiates the coagulation activation in heat stroke and, if so, whether upstream inhibition of coagulation activation through its neutralization may minimize cellular injury and organ dysfunction. We also examined whether coagulation inhibition influences heat stroke-induced fibrinolytic and inflammatory responses. DESIGN: Randomized controlled study. SETTING: Comparative Medicine Department, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia. SUBJECTS: Baboons (Papio Hamadryas). INTERVENTIONS: Twelve anesthetized baboons assigned randomly to recombinant nematode anticoagulant protein c2, a powerful inhibitor of tissue factor/factor VIIa-dependent coagulation (n = 6), or a control group (n = 6) were heat-stressed in a prewarmed neonatal incubator at 44-47°C until systolic blood pressure fell <90 mm Hg, signaling the onset of severe heat stroke. Recombinant nematode anticoagulant protein c2 was administered as a single intravenous dose of 30 µg/kg body weight at onset of heat stroke. The control group received an equivalent volume of sterile saline intravenously. MEASUREMENTS AND MAIN RESULTS: Heat stroke was associated with coagulation activation and fibrin formation as evidenced by the increased plasma thrombin-antithrombin complexes, endogenous thrombin potential, and D-dimer levels. Recombinant nematode anticoagulant protein c2 induced significant inhibition of thrombin generation and fibrin formation. Inhibition of coagulation in recombinant nematode anticoagulant protein c2-treated animals did not influence either fibrinolysis (assessed by tissue plasminogen activator, plasmin-α2-antiplasmin complexes, and plasminogen activator inhibitor) or the release of pro- and anti-inflammatory cytokines. No difference in markers of cell injury and organ dysfunction was observed between recombinant nematode anticoagulant protein c2-treated and control groups. CONCLUSIONS: Tissue factor/factor VIIa-dependent pathway initiates coagulation activation in induced-heat stroke in the baboon without an effect on fibrinolysis and inflammation. The findings suggest also that coagulation activation is not a prerequisite of cell injury and organ dysfunction.

[The role of recombinant activated factor VII in neuro- surgical and neurocritical patients]. / Rol del factor VII recombinante activado en pacientes neuroquirúrgicos y neu- rocríticos.

Central nervous system haemorrhage is a severe pathology, as a small amount of bleeding inside the brain can result in devastating consequences. Haemostatic agents might decrease the consequences of intra- cranial bleeding, whichever spontaneous, traumatic, or anticoagulation treatment etiology. Proacogulant recombinant activated factor VII (rFVIIa) has been given after central nervous system bleeding, with an off-label indication. In this update, we go over the drug mechanism of action, its role in the treatment of central nervous system haemorrhage and the published evidences regarding this subject. We carried out a literature review concerning the treatment with rFVIIa in central nervous system haemorrhage, neurocritical pathologies and neurosurgical procedures, searching in MEDLINE and in clinical trials registry: http://clinicaltrials.gov (last review September 2010), as well as performing a manual analysis of collected articles, looking for aditional references. The results of randomized clinical trials do not support the systematic administration of rFVIIa for spontaneous intracranial cerebral haemorrhage. In other central nervous system related haemorrhages, the current available data consist on retrospective studies, expert opinion or isolated case reports.

Tissue factor-factor VIIa complex induces epithelial ovarian cancer cell invasion and metastasis through a monocytes-dependent mechanism.

OBJECTIVE: Tumor-associated macrophage infiltration and up-regulation of tissue factor-factor VII (TF-FVIIa) complex have been observed in the peritoneum and stroma of epithelial ovarian cancer (EOC). However, it is not clear how tumor-associated macrophage and TF-FVIIa complex promotes EOC invasion. In the present study, we aimed to determine the mechanism by which interaction of TF-FVIIa and monocytes (MOs) promotes EOC metastasis. METHOD: Matrigel invasion assay was used to analyze the potential of EOC metastasis. Enzyme-linked immunosorbent assay and real-time polymerase chain reaction were used to detect expressions of cytokines and chemokines. Fluorescence-activated cell sorting was used to count the percentage of CD14, CD68, and CD163 of MOs. RESULTS: We found that the TF-FVIIa complex caused dynamic changes in MOs cytokine and chemokine expression. CD14 and CD163 were also upregulated on MOs by TF-FVIIa. Epithelial ovarian cancer cells were cocultured with TF-FVIIa-stimulated MOs, demonstrating increased invasion potential. Interleukin 8 (IL-8) was proposed as the major chemoattractant mediating EOC invasion based on MOs messenger RNA and protein expression profiles. Anti-IL-8 monoclonal neutralizing antibody attenuated EOC cell invasion in a concentration-dependent manner, and tumor necrosis factor α from TF-FVIIa-stimulated MOs was observed to amplify IL-8 production. The following transcription factors in MOs were activated by TF-FVIIa and inhibited by the tissue factor pathway inhibitor: oncogenes HIF-1α, HIF-1ß, Oct I, Oct II, and Egr-1; inflammatory mediators c-Fos and c-Rel; and STAT family members STAT5A and STAT5B. CONCLUSIONS: Our study suggested that the interaction between the TF-FVIIa complex might play a role in mediating EOC invasion and metastasis depending on MOs mechanism.

Role of protease-activated receptor-2 in idiopathic pulmonary fibrosis.

RATIONALE: Activation of the coagulation cascade has been demonstrated in pulmonary fibrosis. In addition to its procoagulant function, various coagulation proteases exhibit cellular effects that may also contribute to fibrotic processes in the lung. OBJECTIVE: To investigate the importance of protease-activated receptor (PAR)-2 and its activators, coagulation factor VIIa (FVIIa)/tissue factor (TF), in the development of idiopathic pulmonary fibrosis (IPF). METHODS: Expression and localization of PAR-2 and its activators were examined in IPF lung tissue. The ability of PAR-2 to mediate various cellular processes was studied in vitro. MEASUREMENTS AND MAIN RESULTS: Expression of PAR-2 was strongly elevated in IPF lungs and was attributable to alveolar type II cells and fibroblasts/myofibroblasts. Transforming growth factor-ß(1), a key profibrotic cytokine, considerably enhanced PAR-2 expression in human lung fibroblasts. FVIIa stimulated proliferation of human lung fibroblasts and extracellular matrix production in a PAR-2-dependent manner, but did not initiate differentiation of fibroblasts into myofibroblasts. PAR-2/FVIIa-driven mitogenic activities were mediated via the p44/42 mitogen-activated protein kinase pathway and were independent of factor Xa and thrombin production. Proproliferative properties of FVIIa were markedly potentiated in the presence of TF and abrogated by TF antisense oligonucleotides. Hyperplastic alveolar type II cells overlying fibroblastic foci were found to be the source of FVII in IPF lungs. Moreover, TF colocalized with PAR-2 on fibroblasts/myofibroblasts in IPF lungs. CONCLUSIONS: The PAR-2/TF/FVIIa axis may contribute to the development of pulmonary fibrosis; thus, interference with this pathway confers novel therapeutic potential for the treatment of IPF.

Effect of glycoPEGylation on factor VIIa binding and internalization.

SUMMARY: Recombinant coagulation factor VIIa (rFVIIa), which is widely used for treatment of bleeding episodes in haemophilia patients with inhibitors, is cleared from the circulation relatively fast with a plasma half-life of 2-4 h. PEGylation is an established and clinically proven strategy for prolonging the circulatory life-time of bio-therapeutic proteins. The aim of this study was to investigate the effect of glycoPEGylation of rFVIIa on rFVIIa binding to its cellular receptors and its subsequent internalization. rFVIIa and glycoPEGylated rFVIIa were labeled with (125)I and the radio-iodinated proteins were used to monitor rFVIIa binding and uptake in endothelial cells and fibroblasts. FVIIa-TF activity at the cell surface was analyzed by a factor X activation assay. Modification of rFVIIa with PEG impaired rFVIIa binding to both endothelial cell protein C receptor and tissue factor (TF) on cell surfaces. The internalization of PEGylated rFVIIa in endothelial cells and fibroblasts was markedly lower compared to the internalization of rFVIIa in these cells. PEGylated rFVIIa was able to activate factor X on TF expressing cell surfaces at a rate similar to that of unmodified rFVIIa when the cells were not subjected to multiple washings to remove the free ligand. General effects such as steric hindrance or changes in electrostatic binding properties of the modified rFVIIa to its receptors are probably responsible for this impairment rather than a loss of specific recognition of the receptors, which could explain near normal activation of factor X by glycoPEGylated rFVIIa on TF expressing cells while its uptake is reduced.

Tissue factor and PAR2 signaling in the tumor microenvironment.

Diverse oncogenic transformations result in the constitutive expression of tissue factor (TF) in cancer cells. The local and systemic activation of the coagulation cascade has long been a recognized hallmark for aggressive cancer, but genetic mouse models and new experimental therapeutics have only recently demonstrated crucial roles for TF initiated cell signaling in the pathogenesis of cancer. On tumor cells, the TF-VIIa binary complex mediates activation of protease activated receptor (PAR) 2 and thereby shapes the tumor microenvironment by inducing an array of proangiogenic and immune modulating cytokines, chemokines, and growth factors. PAR2 also uniquely triggers tumor cell migration by G protein-independent pathways through beta-arrestin scaffolding. Metastatic tumor cells use additional signaling networks of the coagulation cascade by activating PAR1 through thrombin or the ternary TF-VIIa-Xa signaling complex in the vascular and potentially lymphatic system. Selective antagonists of TF-VIIa-PAR2 signaling may be used as antiangiogenic therapy without increasing the risk of bleeding, whereas coagulation and associated signaling pathways on platelets and other host cells may be targeted for therapeutic benefit in advanced cancer and metastatic disease.

Factor VII Deficiency.

The complex formed between the procoagulant serine protease activated factor VII (FVII) and the membrane protein tissue factor, exposed on the vascular lumen upon injury, triggers the initiation of blood clotting. This review describes the clinical picture of FVII deficiency and provides information on diagnosis and management of the disease. FVII deficiency, the most common among the rare congenital coagulation disorders, is transmitted with autosomal recessive inheritance. Clinical phenotypes range from asymptomatic condition, even in homozygotes, to severe disease characterized by life-threatening and disabling symptoms (central nervous system and gastrointestinal bleeding and hemarthrosis), with early age of presentation and the need for prophylaxis. In females, menorrhagia is prevalent and affects two thirds of the patients of fertile age. Although FVII gene mutations are extremely heterogeneous, several recurrent mutations have been reported, a few of them relatively frequent. The study of genotype-phenotype relationships indicates that modifier (environmental and/or inherited) components modulate expressivity of FVII deficiency, as reflected by patients with identical FVII mutations and discordant clinical phenotypes. Several treatment options are available for FVII deficiency: the most effective are plasma-derived FVII concentrates and recombinant activated FVII (rFVIIa). Treatment-related side effects are rare.

Citrate artificially masks the haemostatic effect of recombinant factor VIIa in dilutional coagulopathy.

Most often, thrombelastographic analyses are carried out using citrated blood and re-calcification. However, calcium chelation may affect dynamics of tissue-factor-initiated thrombin generation. The present study investigates the effect of sample anticoagulant on the response of a colloid induced dilutional coagulopathy model to recombinant activated factor VII (rFVIIa) as measured by thrombelastography. Thrombelastographic evaluation of whole blood coagulation activated with minute amounts of tissue factor in a model of in vitro haemodilution with hydroxyethyl starch (HES) 130/0.4 in a prospective laboratory study. Whole blood coagulation was evaluated before and after 30% dilution with HES 130/0.4, and following in vitro addition of rFVIIa to whole blood collected into tubes containing citrate, corn trypsin inhibitor (CTI), and no stabilizers. Haemodilution with HES 130/0.4 induces a coagulopathy characterised by a reduced maximum rate of clot formation and a pronounced reduction in the final clot firmness. With all test mediums investigated, rFVIIa significantly shortened clot initiation phase. In cases of native whole blood and CTI-stabilised whole blood, rFVIIa shortens the clotting time but also demonstrated an acceleration of the maximum velocity of clot formation. When citrate is used as anticoagulants in thrombelastographic clotting assays, these may artificially mask the haemostatic effect of rFVIIa in colloid haemodilution. The effect in vitro of rFVIIa in citrated blood samples may underestimate the haemostatic potential of rFVIIa.

Microarray studies of factor VIIa-activated cancer cells.

Factor VIIa (FVIIa)-induced signal transduction is strongly dependent on cellular surface expression of Tissue Factor (TF) and Protease Activated Receptors (PARs). FVIIa signals primarily through PAR2. This contrasts to thrombin which signals primarily via PAR1 and does so without the assistance of a co-receptor, but by binding to an exosite on PAR1. Various TF:FVII-mediated cellular activities are now well documented and have indicated possible links to inflammation, atherosclerosis, angiogenesis, tissue repair, tumor growth and metastasis. Further knowledge about cellular responses induced by coagulation factors has been obtained by gene-expression profiling of MDA-MB-231 cells stimulated with FVIIa or alternatively with PAR1 or PAR2 agonist peptides. These studies and qPCR measurements of the transcription of selected genes in these and other carcinoma cell lines have provided new information about gene expression induced by PAR activation, the gene repertoire induced by TF:FVIIa via PAR2, and how it differs from that induced via PAR1 by thrombin.

Factor VIIa binding to endothelial cell protein C receptor.

Recent studies have shown that factor VIIa (FVIIa) binds specifically to endothelial protein C receptor (EPCR), a known cellular receptor for protein C and activated protein C, on the endothelium. The formation of FVIIa:EPCR complexes neither supports the activation of coagulation nor modulates tissue factor-initiated coagulation. However, FVIIa interaction with EPCR, particularly at pharmacological concentrations of FVIIa, may impair EPCR-dependent protein C activation and activated protein C-mediated cell signaling by competing directly with them for binding to EPCR. FVIIa binding to EPCR may also contribute to FVIIa clearance. This review summarizes recent data on FVIIa interaction with EPCR and discusses potential physiological significance and consequences of the interaction.

Factor VIIa interaction with tissue factor and endothelial cell protein C receptor on cell surfaces.

Factor VIIa (FVIIa) is the enzyme that triggers activation of the clotting cascade that eventually leads to fibrin deposition and platelet activation. Association of FVIIa with its cellular receptor, tissue factor (TF), which greatly increases FVIIa enzymatic activity, is essential for the effective initiation of the coagulation pathway. FVIIa also complexes with endothelial cell protein C receptor (EPCR), but this association does not increase the enzymatic activity of FVIIa. This article reviews current knowledge of FVIIa interaction with TF and EPCR on cell surfaces with a specific focus on how these interactions may contribute to FVIIa and TF clearance, thereby regulating TF-FVIIa activity.

Tissue factor and protease-activated receptor signaling in cancer.

The activation of the coagulation cascade in the tumor microenvironment is a key feature of advanced malignancies. On tumor cells, tissue factor (TF) plays a central role to initiate cross-talk through the release of procoagulant microparticles or through direct, protease-activated receptor (PAR)-mediated cell signaling that leads to the production of soluble cytokines and angiogenic growth factors. In addition, the hemostatic system in the host compartment sustains crucial circuits that promote metastasis and support tumor growth and angiogenesis. Experimental tumor and genetic models have defined specific pathways that are supported by tumor cell and host TF and have identified potential therapeutic modalities to specifically interrupt TF signaling in tumor biology without impairment of hemostatic functions.

Phenotypic correction of hemophilia A by ectopic expression of activated factor VII in platelets.

Platelets are receiving much attention as novel target cells to secrete a coagulation factor for hemophilia gene therapy. In order to extend the application of platelet-directed gene therapy, we examined whether ectopic expression of activated factor VII (FVIIa) in platelets would result in an efficient bypass therapy to induce sufficient thrombin generation on platelet surfaces in mice with hemophilia A. Transduction of bone marrow cells with a simian immunodeficiency virus (SIV)-based lentiviral vector harboring the platelet-specific GPIb alpha promoter resulted in efficient transgene expression in platelets. FVIIa antigen was expressed in platelets by this SIV system; FVII transgene products were found to localize in the cytoplasm and translocate toward the sub-membrane zone and cell surface after activation. Although FVII antigen levels in platelets did not reach the therapeutic levels seen with FVIIa infusion therapy, whole-blood coagulation, as assessed by thromboelastography, was significantly improved in mice with hemophilia A. Further, we observed correction of the bleeding phenotype in mice with hemophilia A after transplantation, even in the presence of FVIII-neutralizing antibodies. Our results demonstrate that FVIIa-expressing platelets can strengthen hemostatic function and may be useful in treating hemophilia and other inherited bleeding disorders. These findings are comparable to the proven therapeutic effects of FVIIa infusion.