Tissue factor-dependent factor VIIa signaling

Tissue factor (TF) is known primary as a cofactor for factor VIIa-mediated triggering of blood coagulation, which proceeds in a cascade of extracellular reactions. Recent investigations have, however, revealed that intracellular activities can also be induced by the proteolytic activity of factor VIIa bound to cell surface TF. Factor VIIa signal transduction has thus been reported to induce mobilization of intracellular Ca(2+) stores and p44/p42 MAPK phosphorylation and to result in expression of specific genes, which presumably affects a number of cellular functions. The factor VIIa-induced signal transduction is independent of the presence of the TF cytoplasmic domain and it is distinctly different from signaling involving presently known protease-activated receptors (PARs) including receptors for thrombin and factor Xa. This short review summarizes recent advances in our understanding of TF-dependent factor VIIa signaling.     

The effects of shear rate on the enzymatic activity of the tissue factor-factor VIIa complex

A novel enzyme reactor for studying phospholipid-dependent reactions was used to explore the effects of flow on tissue factor (TF)-initiated coagulation. Capillary tubes (0.27 mm i.d.) were coated with a phospholipid bilayer containing TF, a transmembrane protein that is an essential cofactor for factor VII. Production of factor Xa exiting the tube was monitored with time during perfusion of the capillary with factor X (50 to 1500 nM) in the presence of factor VIIa (10 nM). Steady-state production of factor Xa as a function of [FX] was determined by chromogenic assay (Spectrozyme Xa) for a range of wall shear rates (25 to 3000 sec-1). Diffusion was found to play a major limiting role in FXa production for TF:30% phosphatidylserine (PS)/70% phosphatidylcholine (PC) surfaces. In contrast, TF/PC surfaces slowed the reaction sufficiently to enter a kinetically controlled regime where shear fluid had little effect on Km. In contrast with classical enzyme kinetic theory there was a three-fold increase in Vmax as shear increased from 25 to 300 sec-1. This finding implies a direct effect of shear on the kinetics of factor X activation by TF/FVIIa. The perfusion system is simple to use and offers the potential for studying the role of flow on a wide variety of enzymatic reactions related to coagulation.     

Tissue factor- and factor X-dependent activation of protease-activated receptor 2 by factor VIIa

Protease-activated receptor 2 (PAR2) is expressed by vascular endothelial cells and other cells in which its function and physiological activator(s) are unknown. Unlike PAR1, PAR3, and PAR4, PAR2 is not activatable by thrombin. Coagulation factors VIIa (FVIIa) and Xa (FXa) are proteases that act upstream of thrombin in the coagulation cascade and require cofactors to interact with their substrates. These proteases elicit cellular responses, but their receptor(s) have not been identified. We asked whether FVIIa and FXa might activate PARs if presented by their cofactors. Co-expression of tissue factor (TF), the cellular cofactor for FVIIa, together with PAR1, PAR2, PAR3, or PAR4 conferred TF-dependent FVIIa activation of PAR2 and, to lesser degree, PAR1. Responses to FXa were also observed but were independent of exogenous cofactor. The TF/FVIIa complex converts the inactive zymogen Factor X (FX) to FXa. Strikingly, when FX was present, low picomolar concentrations of FVIIa caused robust signaling in cells expressing TF and PAR2. Responses in keratinocytes and cytokine-treated endothelial cells suggested that PAR2 may be activated directly by TF/FVIIa and indirectly by TF/FVIIa-generated FXa at naturally occurring expression levels of TF and PAR2. These results suggest that PAR2, although not activatable by thrombin, may nonetheless function as a sensor for coagulation proteases and contribute to endothelial activation in the setting of injury and inflammation. More generally, these findings highlight the potential importance of cofactors in regulating PAR function and specificity.     

Tissue factor and factor VIIa receptor/ligand interactions induce proinflammatory effects in macrophages.

The potential for tissue factor (TF) to enhance inflammation by factor VIIa-dependent induction of proinflammatory changes in macrophages was explored. Purified recombinant human factor VIIa enhanced reactive oxygen species production by human monocyte-derived macrophages expressing TF in vitro. This effect was dose- and time-dependent, ligand- and receptor-specific, and independent of other coagulation proteins. This receptor/ligand binding induced phospholipase C-dependent intracellular calcium fluxes. Transfection studies using a human monocyte-derived cell line (U937) demonstrated that an intact intracytoplasmic domain of TF is required for factor VIIa-induced intracellular calcium fluxes. The capacity of TF to enhance proinflammatory functions of rabbit peritoneal-elicited macrophages (production of reactive oxygen species and expression of major histocompatibility complex class II and cell adhesion molecules) was demonstrated in vivo by treatment with an anti-TF antibody. These data demonstrate that, in addition to its role in activation of coagulation, TF can directly augment macrophage activation. These effects are initiated by binding factor VIIa and are independent of other coagulation proteins. These studies provide the first demonstration of a direct proinflammatory role for TF acting as a cell-signaling receptor.     

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.     

Tissue deformation spatially modulates VEGF signaling and angiogenesis

Physical forces play a major role in the organization of developing tissues. During vascular development, physical forces originating from a fluid phase or from cells pulling on their environment can alter cellular signaling and the behavior of cells. Here, we observe how tissue deformation spatially modulates angiogenic signals and angiogenesis. Using soft lithographic templates, we assemble three-dimensional, geometric tissues. The tissues contract autonomously, change shape stereotypically and form patterns of vascular structures in regions of high deformations. We show that this emergence correlates with the formation of a long-range gradient of Vascular Endothelial Growth Factor (VEGF) in interstitial cells, the local overexpression of the corresponding receptor VEGF receptor 2 (VEGFR-2) and local differences in endothelial cells proliferation. We suggest that tissue contractility and deformation can induce the formation of gradients of angiogenic microenvironments which could contribute to the long-range patterning of the vascular system.     

Antiapoptotic effect of coagulation factor VIIa

Binding of factor VIIa (FVIIa) to its cellular receptor tissue factor (TF) was previously shown to induce various intracellular signaling events, which were thought to be responsible for TF-mediated biologic effects, including angiogenesis, tumor metastasis, and restenosis. To understand the mechanisms behind these processes, we have examined the effect of FVIIa on apoptosis. Serum deprivation-induced apoptosis of BHK(+TF) cells was characterized by apoptotic blebs, nuclei with chromatin-condensed bodies, DNA degradation, and activation of caspase 3. FVIIa markedly decreased the number of cells with apoptotic morphology and prevented the DNA degradation as measured by means of TdT-mediated dUTP nick end labeling (TUNEL). The antiapoptotic effect of FVIIa was confirmed by the observation that FVIIa attenuated caspase 3 activation. FVIIa-induced antiapoptotic effect was dependent on its proteolytic activity and TF but independent of factor Xa and thrombin. FVIIa-induced cell survival correlated with the activation of Akt and was inhibited markedly by the specific PI3-kinase inhibitor, LY294002. Blocking the activation of p44/42 mitogen-activated protein kinase (MAPK) by the specific mitogen-induced extracellular kinase (MEK) inhibitor, U0126, impaired modestly the ability of FVIIa to promote cell survival. In conclusion, FVIIa binding to TF provided protection against apoptosis induced by growth factor deprivation, primarily through activation of PI3-kinase/Akt pathway, and to a lesser extent, p44/42 MAPK pathway.     

Safety profile of recombinant factor VIIa

Recombinant factor VIIa (rFVIIa; NovoSeven®, Novo Nordisk, Bagsvaerd, Denmark) has been used for many years in the successful management of bleeding episodes in patients with hemophilia and inhibitors. More recently, rFVIIa has also shown considerable success as a hemostatic agent in trauma and surgery patients without pre-existing coagulopathy. Despite extensive and varied usage of rFVIIa, the incidence of serious adverse events associated with its use is less than 1%; however, there remain concerns regarding the agent’s potential to induce thrombosis. This paper will review the safety profile of rFVIIa by examining existing clinical evidence, and will demonstrate that the isolated thrombotic events reported following rFVIIa treatment are due primarily to an improvement in the coagulation mechanism rather than rFVIIa treatment per se. The demonstrated safety of rFVIIa is probably due to its localization to injured areas of the vascular tree by binding to tissue factor (TF) and activated platelets at the bleeding site, thus avoiding systemic activation of coagulation. Finally, those situations in which rFVIIa therapy may not be safe, such as disseminated intravascular coagulation (DIC) and sepsis, will also be discussed.     

Tissue factor trafficking in fibroblasts: involvement of protease-activated receptor-mediated cell signaling

Tissue factor (TF) is the cellular receptor for clotting factor VIIa (FVIIa), and the formation of TF-FVIIa complexes on cell surfaces triggers the activation of the coagulation cascade and the cell signaling. Our recent studies have shown that a majority of TF resides in various intracellular compartments, predominantly in the Golgi, and that FVIIa binding to cell surface TF induces TF endocytosis and mobilizes the Golgi TF pool to translocate it to the cell surface. This present study is aimed to elucidate the mechanisms involved in TF endocytosis and its mobilization from the Golgi. Activation of protease-activated receptor 1 (PAR1) and PAR2 by specific peptide agonists and proteases, independent of FVIIa, mobilized TF from the Golgi store and increased the cell surface expression of TF. Blocking PAR2 activation, but not PAR1, with neutralizing antibodies fully attenuated the FVIIa-induced TF mobilization. Consistent with these data, silencing the PAR2 receptor, and not PAR1, abrogated the FVIIa-mediated TF mobilization. In contrast to their effect on TF mobilization, PAR1 and PAR2 activation, in the absence of FVIIa, had no effect on TF endocytosis. However, PAR2 activation is found to be critical for the FVIIa-induced TF endocytosis. Overall the data herein provide novel insights into the role of PARs in regulating cell surface TF expression.     

Factor VIIa bound to endothelial cell protein C receptor activates protease activated receptor-1 and mediates cell signaling and barrier protection

Recent studies have shown that factor VIIa (FVIIa) binds to the endothelial cell protein C receptor (EPCR), a cellular receptor for protein C and activated protein C, but the physiologic significance of this interaction is unclear. In the present study, we show that FVIIa, upon binding to EPCR on endothelial cells, activates endogenous protease activated receptor-1 (PAR1) and induces PAR1-mediated p44/42 mitogen-activated protein kinase (MAPK) activation. Pretreatment of endothelial cells with FVIIa protected against thrombin-induced barrier disruption. This FVIIa-induced, barrier-protective effect was EPCR dependent and did not involve PAR2. Pretreatment of confluent endothelial monolayers with FVIIa before thrombin reduced the development of thrombin-induced transcellular actin stress fibers, cellular contractions, and paracellular gap formation. FVIIa-induced p44/42 MAPK activation and the barrier-protective effect are mediated via Rac1 activation. Consistent with in vitro findings, in vivo studies using mice showed that administration of FVIIa before lipopolysaccharide (LPS) treatment attenuated LPS-induced vascular leakage in the lung and kidney. Overall, our present data provide evidence that FVIIa bound to EPCR on endothelial cells activates PAR1-mediated cell signaling and provides a barrier-protective effect. These findings are novel and of great clinical significance, because FVIIa is used clinically for the prevention of bleeding in hemophilia and other bleeding disorders.     

Binding of factor VIIa to tissue factor permits rapid antithrombin III/heparin inhibition of factor VIIa

Because free factor VIIa is inactivated only very slowly by a plasma concentration of antithrombin III (AT III) even in the presence of heparin, it has been assumed that AT III plays no significant role in regulating the initiation of tissue factor-dependent blood coagulation. However, in the present study, we present evidence that factor VIIa bound to tissue factor, unlike free factor VIIa, is readily inactivated by AT III in the presence of heparin. In a reaction mixture containing calcium ions and approximately equimolar concentrations of relipidated tissue factor (8.9 nmol/L) and factor VIIa (10 nmol/L), AT III (100 micrograms/mL) plus heparin (1 U/mL) inhibited 50% of the factor VIIa coagulant activity of the reaction mixture within 5 minutes. AT III/heparin was also shown to inhibit the catalytic activity towards factor X of factor VIIa/tissue factor complexes formed on monolayers of an ovarian carcinoma cell line (OC-2008) that constitutively expresses surface membrane tissue factor. AT III, even in the absence of exogenously added heparin, substantially inhibited the functional activity of factor VIIa/cell surface tissue factor complexes on intact monolayers. AT III alone and AT III/heparin, to a greater extent, also inhibited factor VIIa on "nonfunctional" factor VIIa/tissue factor complexes on intact monolayers, with resultant inhibition of their expression of factor VIIa/tissue factor catalytic activity toward factor X after cell lysis. The potential physiologic significance of these findings is discussed.