Tissue Factor–Factor VIIa Signaling
http://www.100md.com
《动脉硬化血栓血管生物学》
From the Biomedical Research Division, The University of Texas Health Center at Tyler, Tex.
Correspondence to L. Vijaya Mohan Rao, PhD, Biomedical Research, The University of Texas Health Center at Tyler, 11937 US Highway 271, Tyler, TX 75708. E-mail vijay.rao@uthct.edu
Abstract
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.
Recent studies show that tissue factor–factor VIIa, whose primary function is to initiate the clotting cascade, transduces cell signaling in various cell types. This brief review summarizes recent literature on potential mechanisms by which tissue factor–factor VIIa activates cell signaling, and how tissue factor–factor VIIa-induced cell signaling may affect various pathophysiological processes.
Key Words: tissue factor ? factor VIIa ? protease activated receptors ? cell signaling
Introduction
The close link between coagulation and various diseases, such as sepsis, atherosclerosis, and tumor metastasis, suggests a complex interplay between the clotting cascade and disease progression. Numerous studies have demonstrated that coagulation proteases can function like hormones to regulate cellular behavior. For example, thrombin, the principal protease generated during coagulation, has been shown to activate platelets and regulate the behavior of other cells by transmitting signals via activation of G protein-coupled protease activated receptors (PARs). Although there has been initial skepticism on whether other clotting proteases can also activate PARs, recent studies provide convincing evidence that many proteases involved in clotting can indeed activate PARs and regulate cellular behaviors at physiological concentrations. One such protease, FVIIa, the physiological initiator of the coagulation cascade, has received much attention lately. The focus of the present article is to review briefly recent developments in tissue factor (TF)–VIIa proteolytic activity-mediated cell signaling.
TF–Factor VIIa
TF is a transmembrane cellular receptor for FVII/FVIIa. Binding of plasma FVII/FVIIa to TF triggers the coagulation cascade, which leads to thrombin generation that subsequently stimulates platelet activation and cleaves fibrinogen. Under normal conditions, TF is constitutively expressed in many cell types, including fibroblasts and pericytes in and surrounding blood vessel walls, but is not expressed in blood cells or the endothelial cells that line blood vessels.1,2 However, under certain pathological conditions, such as sepsis and cancer, monocytes3 and endothelial cells4 express TF, although the latter finding has not been confirmed by others.5 Thus, blood vessel wall injury or certain disease conditions permit FVII/FVIIa interaction with TF on cell surfaces. The activation of TF-induced coagulation pathway not only leads to fibrin formation but also contributes to vascular remodeling, which is caused by growth factors secreted by activated platelets, as well as the intermediary products factor Xa and thrombin that promote vascular smooth cell proliferation6,7 and alter endothelium.8 TF expression has been linked directly to various pathophysiological processes, such as development, inflammation, and tumor metastasis.9–11
PARs
Certain proteases control cellular functions via signaling through specific membrane receptors known as PARs, members of a larger family of 7 transmembrane cell surface receptors that mediate cell activation via G-proteins. At present, there are 4 known PARs: PAR1, PAR2, PAR3, and PAR4. PAR1, the prototypic family member, is cleaved primarily by thrombin, but can also be cleaved by other proteases, such as FXa,12 activated protein C (APC),13 plasmin,14 and FVIIa.15 PAR3 and PAR4 are also cleaved by thrombin, whereas PAR2 is cleaved by the trypsin-like enzymes, FXa16 and FVIIa.15 Although the mechanism of PARs activation was initially established for thrombin and PAR1, the mechanism is similar for the other PARs. In short, the protease cleaves the receptor at a specific site in the amino-terminal extracellular domain, leading to the unmasking of a new N-terminus. The new N-terminus then acts as a tethered ligand, binding intramolecularly to the body of the receptor to initiate transmembrane signaling. In fact, synthetic peptides mimicking the tethered ligand can activate the receptor independent of its cleavage. A number of excellent reviews provide details of the mechanism of activation of PARs and define the role for these receptors in vivo.17–19
TF–VIIa-Induced Signaling
Because aberrant expression of TF is associated with various diseases and TF is structurally similar to members of the class 2 cytokine receptor family,20 there has been a great interest in examining the role of TF in cell signaling. The first evidence for TF–VIIa-induced signaling came from studies in which FVIIa binding to TF was shown to induce intracellular Ca2+ oscillations in a number of TF-expressing cells.21,22 This FVIIa-induced calcium signaling required the binding of catalytically active FVIIa to TF but did not require the presence of TF cytoplasmic domain. Although cells that express TF, either inducibly or constitutively, responded to FVIIa binding, the fraction of cells that released Ca2+ and the extent to which Ca2+ was released varies among different cell types.23 Interestingly, some cell lines, such as HK-2 (a kidney cell line that constitutively expresses TF) and CHO cells stably transfected with TF, failed to respond to FVIIa, despite abundant expression of TF on their surfaces.24
Work from other laboratories demonstrated that FVIIa interaction with TF on a variety of cells, including fibroblasts, epithelial cells, and endothelial cells, activated multiple signaling pathways. Poulsen et al25 reported first that FVIIa binding to BHK cells that are stably transfected with human TF [BHK(TF)] resulted in a transient activation of p44/42 MAPK. Further studies showed that the FVIIa-induced p44/42 MAPK activation is dependent on proteolytically active FVIIa, but independent of the TF cytoplasmic domain.26 FVIIa is shown to activate p44/42 MAPK in other cell types that express TF, such as fibroblasts27 and keratinocytes,28 but the response is not as robust as that observed in BHK(TF) cells. Additionally, FVIIa treatment of keratinocytes also increased the phosphorylation of key components of the other 2 MAPK pathways, p38 and C-Jun N-terminal kinase (JNK).24 Versteeg et al29 demonstrated that FVIIa stimulates a signaling pathway in fibroblasts (A14 cells), leading to the activation of the Src-like family members c-Src, Lyn, and Yes, and subsequently PI3-kinase, which then induces the stimulation of p44/42 MAPK, c-Akt/protein kinase B, and the small GTPases Rac and Cdc42. This group also showed that FVIIa-induced p44/42 MAPK activation is mediated via p21 Ras activation in BHK(TF) and HaCaT keratinocytes.30 In HaCaT cells, FVIIa has been shown to phosphorylate PYK2,31 a kinase that has been implicated in the regulation of MAP kinase activation.32 In addition to activating the p21 ras/MAPK pathway, FVIIa has also been shown to induce STAT5 phosphorylation via Jak2 activation in BHK(TF) cells33 and to stimulate the protein synthesis machinery via activation of p70/p85s6K, p90RSK, and eventually eukaryotic initiation factor eIF-4E.34 Although the described data provide convincing evidence that TF–VIIa activates multiple signaling pathways that could affect various cellular processes, one should exercise caution in extrapolating these data because the majority of these data were derived from a single cell line, BHK(TF).
To address how TF-mediated cell signaling could potentially contribute to various pathophysiological conditions, several groups have focused on investigating TF–VIIa-induced alterations in gene expression. Examination of specific gene transcripts, whose products were believed to be pathophysiologically relevant to diseases associated with aberrant expression of TF, revealed that exposure of TF-expressing cells (fibroblasts, tumor cells, or keratinocytes) to FVIIa led to increased expression of vascular endothelial growth factor (VEGF),35 uPAR,36 Egr-1,24 and IL-8.37,38 Global analysis of TF–VIIa protease-induced signaling on the transcriptional machinery revealed that FVIIa binding to TF on fibroblasts or keratinocytes alters the expression of a few select genes. One of the upregulated transcripts observed in differential display polymerase chain reaction was identified as a poly(A) polymerase, whose product plays an important role in the processing of mRNA. Microarray analysis of fibroblasts briefly exposed to FVIIa revealed that FVIIa upregulates the expression of Cyr61 (CCN1) and connective tissue growth factor (CCN2).39 CCN1 and CCN2 are extracellular matrix signaling proteins that were recently shown to regulate a myriad of cellular functions, such as cell adhesion, proliferation, migration, and tumor metastasis.40 Using low-density cDNA arrays, Camerer et al28 showed that FVIIa interaction with TF on keratinocytes upregulates several genes that are relevant to the wound repair process.
TF–VIIa Activation of a PAR
Because TF–VIIa-induced signaling requires catalytically active FVIIa and is independent of the TF cytoplasmic tail, it has been hypothesized that TF–VIIa transmits cell signaling via PAR activation. However, until recently there was confusion about whether TF–VIIa mediated the cell signaling via 1 of the 4 known PARs or involved the activation of a novel PAR. The lack of heterologous desensitization (ie, failure of PAR agonists to abolish the response induced by subsequently added VIIa or vice versa), the differences between PAR agonists and FVIIa for their ability to induce Ca2+ release and p44/42 MAPK activation, and the lack of FVIIa-induced response in cells that express TF and the known PARs indicated that FVIIa may induce intracellular signaling via activation of a novel PAR or requires, in addition to a known PAR, an additional cell surface component.21,24,26,39,41 However, a recent study by Camerer et al15 showed that FVIIa induces Ca2+ release in Xenopus oocytes transfected with TF together with PAR1 or PAR2, but not PAR3 and PAR4. Similarly, transfection of lung fibroblasts from PAR1-deficient mice with TF and PAR2 conferred the FVIIa-induced response.15 These data suggest that TF–VIIa activates PAR2 and, to a lesser degree, PAR1. This conclusion is further supported by the observation that FVIIa can induce p44/42 MAPK activation in CHO cells transfected with TF and PAR2 but not in CHO cells expressing TF alone.42 Additionally, recent data38 demonstrate that specific antibodies against PAR2 but not PAR1 block FVIIa-induced IL-8 gene expression and cell migration in breast carcinoma cells. Similarly, PAR2 antibodies were shown to block TF–VIIa-induced smooth muscle cell migration.43 Together, these data suggest that TF–VIIa transmits cell signaling via activation of PAR2 (Figure 1). However, this does not mean that TF–VIIa cannot transmit signals via receptors other than PAR2.
Figure 1. A schematic representation of TF–VIIa protease-induced signaling. TF–VIIa activates PAR2 and the ternary complex of TF–VIIa–Xa activates both PAR1 and PAR2. Activation of PAR2-specific signaling pathway may lead to phosphorylation of TF cytoplasmic tail. Phosphorylation of TF cytoplasmic domain releases its negative regulatory control of PAR2-mediated signaling.
At present, it is unclear why FVIIa fails to induce calcium signaling in a number of cell types, including fibroblasts, epithelial cells, and tumor cells that express abundant TF and one or more PARs (PAR1 and PAR2)27,38,41 (and also unpublished data of the authors), and respond robustly to FVIIa stimulation by activating p44/42 MAPK25,41 or gene expression.39 In fact, failure to induce Ca2+ release and the lack of the desensitizing effect of FVIIa on PAR1 and PAR2 agonist-induced Ca2+ release lead to a conclusion, which in retrospective is not well-justified, that TF–VIIa-induced intracellular signaling is not mediated via known PARs but may involve proteolytic cleavage of an unidentified member of the PAR family.41 Why does activation of PAR2/PAR1 by TF–VIIa, in contrast to their activation by thrombin, trypsin, or PAR agonist peptides, fail to induce Ca2+ mobilization? One possible explanation may be an inefficient, but physiologically important, cleavage of PAR2/PAR1 by TF–VIIa. Earlier studies44 have shown that the magnitude of the PAR1-mediated protease response is determined by both the rate and extent of receptor cleavage. A low concentration of protease or in the presence of an inefficient protease, only a limited number of receptors would be activated. Although a limited number of activated receptors could transduce a signal, a measurable response requires activation of a minimum number of receptors at a certain rate.19 Additionally, the rate and extent of receptor activation required to elicit a specific response may be dependent on the response that is measured. For example, when receptor cleavage correlates with phosphoinositide hydrolysis, IP3 formation is proportional to the absolute amount of cleaved receptor, but the subsequent increase in cytosolic Ca2+ occurs only if IP3 is generated quickly enough to accumulate.44 However, this reasoning may not fully explain the phenomenon described given that FVIIa (10 nM), trypsin (1 nM), and PAR2 peptide agonist (1 μmol/L) treatments all result in a similar rate of IP3 hydrolysis, yet only trypsin and PAR2 peptide agonist, and not FVIIa, produced a clear increase in Ca2+ release (unpublished data of authors).
Although the TF–VIIa complex is sufficient to induce cell signaling, it is unclear whether this binary complex functions as an efficient signaling unit in vivo. In many of the studies described here, high concentrations (10 to 100 nM) of FVIIa were required to obtain a measurable signaling response.15,21,26,42 It is likely that such high concentrations of FVIIa are needed to saturate TF rapidly, which may be essential for measuring the signaling response using a short-term assay, such as Ca2+ release or MAPK activation. In fact, low concentrations of FVIIa (5 to 10 nM) are shown to be capable of producing a pronounced response when signaling is analyzed using a long-term assay, such as gene expression28,38,39 However, in circulation most of the FVII is in zymogen form. FVIIa concentration in the plasma is 1% or less (100 pM) of the total circulating FVII (10 nM).45,46 Thus, it is crucial to show that traces of FVIIa in FVII are sufficient to induce cell signaling. However, it is pertinent to note here that because FVII bound to TF can be autocatalytically converted to FVIIa,47–49 most of the FVII bound to cell surface TF will be converted to FVIIa. Consistent with this notion, we found that plasma concentration of zymogen FVII added to fibroblasts39 and tumor cells38 induced Cyr61 and IL-8 gene expression, respectively, with a slight delay. The delay probably reflects the time required for autoactivation of FVII. When substrate FX is present at plasma concentrations, picomolar concentrations of FVIIa elicit a robust signal in cells expressing TF and PAR2.15 This suggests that the TF–VIIa-generated FXa is capable of signaling independent of thrombin. Riewald and Ruf found that the activation of FX by the TF–VIIa complex resulted in a much more robust signal than that induced by the TF–VIIa complex alone, free FXa, or FXa that was generated in situ by the intrinsic activation complex.42 These data, coupled with additional studies using a unique inhibitor (NAPc2, nematode anticoagulant protein C2) that preserves FXa activity in the complex while inhibiting free FXa and TF–VIIa proteolytic activity, revealed that the transient ternary TF–VIIa–Xa complex is a potent signaling unit in which FXa efficiently activates both PAR1 and PAR2.42 This finding supports the hypothesis that upstream coagulation protease signaling is mechanistically coupled to the initiation of the coagulation pathway. Although our data support the concept that the ternary complex of TF–VIIa–Xa is a more potent activator of PAR2 than the binary TF–VIIa complex, this difference is abolished when TF sites are saturated with FVIIa (Figure 2).38 It is currently difficult to conclude whether the ternary or the binary complex is the primary signaling unit in vivo. It is likely that both complexes play a role in vivo, and which complex is more active may depend on multiple factors, such as the levels of TF and PAR2/PAR1 expression, the availability of FX, and the localization/organization of these components on the cell membrane.
Figure 2. A comparison of efficiency of TF–VIIa-mediated and TF–VIIa–Xa-mediated cell signaling. MDA-MB-231 cells were stimulated with varying concentrations of FVIIa in the presence or absence of substrate FX (175 nM). The signaling was evaluated in either IP3 hydrolysis assay (A) or IL-8 gene expression (B).
Although it is apparent that TF–VIIa elicits cell signaling via activation of PAR2, it is unclear whether other cell membrane components participate in the signaling process. It is interesting to note that in some cases, even a high concentration of FVIIa fails to elicit a signaling response, even in cells expressing both functional TF and PAR2.24,50 It raises a possibility that other cell components may contribute or regulate TF–VIIa-induced cell signaling. In this context, it is important to note that Wiiger and Prydz31 recently demonstrated that the epidermal growth factor receptor is involved in the transduction of the TF–VIIa signal in HaCaT cells. However, the role of epidermal growth factor receptor in TF–VIIa-induced signaling still needs to be confirmed.
TF Cytoplasmic Domain-Dependent Signaling
In heterologous expression studies, the TF cytoplasmic domain is not clearly required for the TF–VIIa-induced cell signaling.24,26,34 However, it is unclear whether TF, as a true receptor, can transmit cell signaling via its cytoplasmic tail, or whether the cytoplasmic domain can regulate the protease-induced signaling. Analysis of human TF protein sequence revealed a consensus sequence for protein kinase C phosphorylation in the cytoplasmic domain, which is shown to be phosphorylated in response to phorbol esters.51 Mody and Carson52 showed, using a synthetic peptide corresponding to residues 245 to 263 of the human TF cytoplasmic domain and glioblastoma cell extract (as a source of kinase activity), that TF cytoplasmic domain is phosphorylated at multiple serine residues. Recent studies show that protein kinase C-dependent phosphorylation of Ser253 enhances subsequent Ser258 phosphorylation by a proline-directed kinase.53 A functional role for the TF cytoplasmic domain was documented in hematogenous metastasis. Deletion of the cytoplasmic domain54,55 or mutation of the cytoplasmic phosphorylation sites Ser253 and Ser25855,56 are shown to reduce the TF-induced metastasis. Abe et al57 showed that the TF cytoplasmic domain, independent of FVIIa, is responsible for the upregulation of VEGF in melanoma cells transfected with TF. However, others failed to confirm this finding.58 Recent studies with transgenic mice that lack the cytoplasmic domain of TF (but have normal coagulant function) indicate that the cytoplasmic domain of TF contributes to NF-B activation, pro-inflammatory cytokine production, and leukocyte recruitment after endotoxin challenge. However, further studies are needed to confirm that the TF cytoplasmic domain is directly responsible for the observed effects. Other studies suggest that the TF cytoplasmic domain contributes to cell signaling indirectly, ie, by modulating TF–VIIa-induced cell signaling. For example, deletion of the TF cytoplasmic domain is shown to impair TF–VIIa protease activity-induced reactive oxygen species production in monocytes. Elegant studies performed recently by Ahamed et al show that TF–VIIa–Xa activation of PAR2 induces TF cytoplasmic domain phosphorylation,59 and this phosphorylation of the TF cytoplasmic domain releases its negative regulatory control of PAR2 signaling-mediated angiogenesis60 (Figure 2). At present, it is unclear whether this regulatory mechanism is specific for endothelial cells and the angiogenic process or if it also plays a role in modulating other PAR2 signaling-mediated events in other cell types.
A Role for TF–VIIa Signaling in Pathophysiology
It is widely accepted that TF, in addition to its role in coagulation, may contribute to various pathophysiological processes. However, it is unclear whether TF–VIIa-induced (or TF–VIIa–Xa-induced) cell signaling contributes directly to these biological processes, or whether cell signaling induced by coagulation proteases generated by TF–VIIa, such as thrombin in concert with an end product fibrin, fully accounts for the altered cellular processes. Recent studies suggest that both mechanisms are involved. The following discussion is limited to the potential role of direct TF–VIIa-induced cell signaling in a select few biological processes (Figure 3). For discussion on the role of TF in development, the reader is referred to a recent review.11
Figure 3. TF–VIIa signaling mediated cellular effects and their contribution to various pathophysiological processes (see text for details).
Inflammation
It is well-established that inflammatory mediators activate coagulation by inducing TF expression on blood mononuclear cells and probably on vascular endothelium. In turn, expression of TF on these cells appears to regulate the inflammatory response. The observation that during sepsis the TF-initiated coagulation pathway induces a lethal inflammatory escalation even when fibrin formation and microthrombosis are effectively blocked via decreased thrombin generation61–65 suggests that TF–VIIa cell signaling induces a pro-inflammatory response. Additionally, inhibition of the TF–VIIa complex during septicemia significantly reduces the inflammatory response, as measured by decreased IL-6 and IL-8 plasma levels and reduced inflammatory changes in the lung, such as neutrophil infiltration and edema.63,66 In vitro studies67 also indicate that TF plays an important role in reverse transmigration of mononuclear phagocytes, a process that occurs during the resolution of acute inflammation. FVIIa binding to TF has been shown to augment the macrophage pro-inflammatory functions, such as the production of reactive oxygen species and the expression of major histocompatability complex class II and cell adhesion molecules, both in vivo and in vitro.68 Furthermore, recent studies using the mouse model of endotoxemia have shown that genetically modified mice expressing low levels of TF exhibited reduced IL-6 induction and increased survival compared with control mice.69 Although a deficiency of either PAR1 or PAR2 has no effect on inflammation or survival, a combination of thrombin inhibition and PAR2 deficiency reduces both inflammation and mortality similar to that observed in the low-TF mice.69 These data suggest that PAR1 and PAR2 may have partially redundant roles and contribute to the link between coagulation and inflammation. Together, these data imply that TF–VIIa-induced cell signaling via PAR2 may contribute to pro-inflammation. However, it is difficult to conclude from these results whether the pro-inflammatory signaling unit is TF–VIIa, TF–VIIa–Xa, or FXa, given that all of them are capable of activating PAR2. Furthermore, the reduced lipopolysaccharide-induced IL-6 expression and enhanced survival in the low-TF mice may be unrelated to the defect in TF–VIIa protease-induced signaling given that similar effects were observed in mice expressing TF that lacks the cytoplasmic domain and has normal coagulant function.70 However, one should exercise caution in interpreting the data from the latter studies because these studies were performed in mice with a mixed genetic background, which likely leads to a high degree of variability.
Tumor Angiogenesis
Hemostasis and angiogenesis are interrelated processes. Proteins generated by the hemostatic system are known to have regulatory effects on angiogenesis.71 TF-induced coagulation can indirectly support angiogenesis in several ways, such as the release of positive and negative regulators from activated platelets, thrombin signaling via endothelial cell PAR1, and the generation of provisional fibrin-rich matrix. Whether TF–VIIa signaling plays a direct role in the regulation of angiogenesis is not entirely clear. Overexpression of TF in tumor cells leads to the upregulation of the pro-angiogenic factor VEGF and the downregulation of the anti-angiogenic protein thrombospondin-1.72 However, Bromberg et al found no correlation between TF expression and VEGF.56 Recent studies have shown that TF–VIIa induces IL-8 production via PAR2 signaling, and the secreted IL-8 is capable of stimulating tumor cell migration and invasion in an autocrine fashion.38 IL-8 secreted by tumor cells was shown previously to regulate angiogenesis directly by enhancing endothelial cell survival, proliferation, and matrix metalloproteinase production.73 Surprisingly, although many cancer cell lines express functional PAR1, PAR2, and TF, these cells respond to either thrombin or FVIIa, but not to both in increasing IL-8 (unpublished data of authors). This observation suggests that other cell components, either on the cell surface or within the cell, provide further specificity for the protease-induced signaling.
In cancer cells (unpublished data of the authors) and fibroblasts,39 TF–VIIa signaling upregulates the expression of CCN1, a novel matrix signaling protein that is a ligand to integrin v?3 on endothelium.74 Integrin v?3, an adhesion receptor known to be involved in signaling, regulates a number of cellular processes, including angiogenesis and tumor metastasis. The stimulatory effects of CCN1 on cell proliferation, migration, and survival via its interaction with various integrins are thought to be responsible for its role in angiogenesis and tumorigenesis.75 In addition to CCN1 and IL-8, TF–VIIa-induced cell signaling may also upregulate a number of other gene products, including uPAR,36 which plays a regulatory role in angiogenesis. Microarray analyses of MDA-MB-231 breast carcinoma cells exposed to FVIIa and a control vehicle show that FVIIa induces a set of genes whose products play a role in various steps of angiogenesis and tumor growth. The gene products include chemokines, cytokines, growth factors, cell adhesion proteins, and proteins involved in cell cycle control, apoptosis, and inflammation (unpublished data). Together, these data support the hypothesis that TF–VIIa cell signaling may play an important role in angiogenesis regulation (Figure 4).
Figure 4. TF–VIIa signaling and angiogenesis. Binding of FVIIa to tumor cell TF activates a genetic program, which leads to upregulation of various chemokines, cytokines, and growth factors, including IL-8, CCN1, and VEGF. These secreted cytokines and growth factors from tumor cells promote endothelial cell proliferation and migration, leading to angiogenesis.
Recent studies indicate a complex role for TF in angiogenesis. Belting et al60 show that genetic deletion of the TF cytoplasmic domain enhances PAR2-dependent angiogenesis, presumably in synergy with platelet-derived growth factor BB. In neonatal mice, the diameter of the superficial vascular plexus of TF–cytoplasmic domain-deleted mice was twice that observed in wild-type mice, indicating that the TF cytoplasmic tail negatively regulates in vivo angiogenesis during postnatal development. Furthermore, ocular tissues from diabetic patients display a colocalization of PAR2 and phosphorylated TF specifically on neovasculature. Overall, these observations suggest that phosphorylation of the TF cytoplasmic domain releases its negative regulatory control of angiogenesis. The role of the TF cytoplasmic domain as a negative regulator of physiological process has been further supported by the recent observation that TF expression suppresses 3?1-dependent migration on laminin 5, an effect that is reversed by PAR2-dependent phosphorylation of the TF cytoplasmic domain.76 Although these data support nonhemostatic roles for TF in angiogenesis and tumor metastasis, they do not fully explain the earlier studies that demonstrated that the TF–cytoplasmic domain contributes to cell migration,77 VEGF production,57 and tumor metastasis.54,55 The identified link between PAR2–TF and TF–integrins add yet another facet to the complex regulation of angiogenesis by TF–VIIa signaling.
Tumor Metastasis
The role of TF in metastasis is well-documented. TF expression has been correlated with malignant progression in several types of cancer.78–82 Experiments using mouse models have demonstrated that TF in tumor cells promotes hematogenous metastasis, and both the TF cytoplasmic domain and the TF–VIIa protease activity contribute to this metastasis.54–56,83 It is likely that the principle role for TF–VIIa protease activity in tumor metastasis is to generate thrombin and fibrin, which could play a direct role in tumor metastasis. Nonetheless, there is a possibility that TF–VIIa signaling may also contribute to tumor metastasis because it alters many cellular processes that are associated with tumor metastasis, such as angiogenesis. Furthermore, FVIIa induces the activation of both p42/44 MAPK and protein kinase B pathways, 2 pathways known to inhibit apoptosis.84,85 Resistance to apoptosis is a key factor in the survival of malignant cells. Defects in apoptosis or activation of anti-apoptotic pathways promote tumor growth and survival, which may be linked to tumor metastasis.86,87 Recent studies88,89 have shown that TF–VIIa activates anti-apoptotic signaling pathways in serum-deprived cells, thereby increasing their survival. Additionally, FVIIa was found to inhibit apoptosis induced by the loss of adhesion.88 Therefore, TF–VIIa-induced cell survival, in addition to other TF–VIIa-induced cellular processes, may contribute to tumor growth and metastasis. However, it should be noted that the evidence for the anti-apoptotic effect of FVIIa is limited to cells that were transfected to overexpress TF. Confirmation of the anti-apoptotic effect of FVIIa in tumor cells and/or stromal cells surrounding a tumor is essential before strong mechanistic conclusions of how TF–VIIa contributes to tumor growth and metastasis can be drawn.
Although it is logical to assume that tumor TF contributes to tumor growth and metastasis through TF–VIIa activation of PAR2 and/or PAR1, currently there is no evidence to support this. Both PAR1 and PAR2 are coexpressed in tumor cells and cells surrounding a tumor in the tumor microenvironment.90 Although many studies document the importance of thrombin activation of PAR1 in tumor cells in metastasis,91–97 little is known regarding the role of tumor cell PAR2 in metastasis, let alone the importance of TF–VIIa-induced activation of PAR2. Recently, Shi et al98 showed that both PAR1 and PAR2 are involved in tumor metastasis and PAR2 effects on tumor cell migration and metastasis are thrombin-dependent. At present, it is unclear whether PAR2 in metastasis is activated indirectly by thrombin or directly by a protease with trypsin-like activity, such as FVIIa. Studies addressing the role of PAR1 and PAR2 of host tissues in tumor metastasis show both PAR1 and PAR2 deficiency have no effect on tumor metastasis.99 In contrast, genetic deficiency in platelet production or activation protected mice against metastasis in hematogenous metastasis model system.99 These data raise a valid question whether PAR1 and PAR2 activation in endothelial or inflammatory cells contribute to tumor metastasis.
Wound Healing
The epidermis is a rich source for TF.1 Most wounds to the skin will invariably cause leakage of blood from the damaged blood vessels. This leakage allows the formation of a fibrin clot, which temporarily shields the wound and protects the denuded wound tissues, and it also releases various growth factors and cytokines from degranulating platelets that trigger the repair process immediately after injury.100,101 The blood leakage at the wound site will also allow FVIIa binding to TF on keratinocytes, raising a possibility for a role for TF–VIIa-induced cell signaling in the wound repair. Consistent with this scenario, Camerer et al28 showed that the interaction of FVIIa with TF on human keratinocytes upregulated a number of genes that are shown to be involved in the early steps of wound healing. They include transcription regulators (c-fos, EGR-1, ETR101, BTEB2, c-myc, fra-1, and tristeraproline), growth factors (amphiregulin, hbEGF, CCN2, and FGF-5), pro-inflammatory cytokines (IL-1?, IL-8, LIF, and MIP2), proteins involved in cellular reorganization/migration (RhoE, uPAR, and collagenases 1 and 3), and others (plasminogen activator inhibitor-2, cyclophilin, GADD45, Jagged 1, and prostaglandin E2 receptor). Because FVIIa binding to TF would occur immediately on wounding, TF–VIIa signaling may provide an early signal in the wound repair process. The expression pattern of TF–VIIa-induced genes suggests that TF–VIIa signaling may contribute to various steps in wound healing. TF–VIIa signaling may also play a role in wound healing indirectly through elaboration of gene products that in turn could induce cell signaling appropriate for the wound repair process. In this context, TF–VIIa-induced expression of CCN1 and CCN2 in keratinocytes and fibroblasts are noteworthy.28,39 CCN1 is inducibly expressed in granulation tissue during wound repair and activates genes that play multiple and coordinated roles in the wound healing process, including angiogenesis, inflammation, and extracellular matrix remodeling.102 Similarly, CCN2 contributes to wound healing via selective modulation of fibroblasts proliferation and changes to gene expression.103
Atherosclerosis and Smooth Muscle Cells
It is well-established that exposure of TF to circulating blood on rupture of atherosclerotic plaque plays an important role in the pathogenesis of thrombus formation at sites of plaque rupture, resulting in acute coronary events and myocardial infarction.104–107 Recent studies indicate that TF–VIIa signaling may play a role in the pathogenesis of atherosclerosis. TF–VIIa has been shown to be a strong chemotactic stimulus for smooth muscle cells,108 and overexpression of tissue factor pathway inhibitor in smooth muscle cells was shown to attenuate the TF–VIIa-induced cell migration.109 Consistent with this in vitro observation, overexpression of tissue factor pathway inhibitor was found to attenuate vascular remodeling in a murine model system.110 TF–VIIa-induced cell signaling has also been shown to promote platelet-derived growth factor BB-induced cell migration in fibroblasts.111 Additionally, TF–VIIa-induced cell signaling recently has been shown to lead to smooth muscle cell proliferation.112 Although our recent data113 using fibroblasts support this observation, the proliferative effect of TF–VIIa-induced cell signaling was very modest. Moreover, other studies failed to demonstrate the proliferative effect of TF–VIIa signaling.29,89,114 Therefore, further studies are needed to determine whether TF–VIIa-induced cell proliferation contributes to the pathogenesis of atherosclerosis. Because TF–VIIa-induced cell signaling in various cell types was shown to induce a number of gene products that are relevant to cell proliferation and migration, it is possible that TF–VIIa signaling may also produce similar effects in TF-expressing cells in the atherosclerosis plaques. It is of particular interest to note that TF–VIIa signaling upregulates the expression of CCN1 and CCN2 in fibroblasts.39 Similar to TF expression, CCN1 mRNA is undetectable in normal blood vessels but overexpressed in atherosclerotic lesions, primarily in vascular smooth muscle cells.115 In atherosclerosis, high levels of CCN2 expression is thought to be responsible for extracellular matrix accumulation and thus progression of atherosclerotic lesions.116 Similar to CCN2, CCN1 expression was shown to be upregulated in atherosclerotic lesions of apoE–/– mice117 and human atherosclerotic lesions.118 Because recent studies show extrahepatic synthesis of FVII in human atherosclerotic vessels,119 it is possible that TF–VIIa may induce the expression of CCN1 and CCN2 within the plaque, which could accelerate intimal thickening by promoting proliferation and migration of fibroblasts and smooth muscle cells. Further, removal or retraction of endothelial cells of atherosclerotic plaques would expose CCN1 and CCN2 in the underlying subendothelial matrix to which activated platelets and monocytes could adhere.117,120 This, in combination with TF–VIIa-induced fibrin clot, could lead to an acute arterial occlusion on rupture of the atherosclerotic plaque. Paradoxically, increased expression of CCN2 could also play a beneficial role. For example, increased expression of CCN2 along the fibrous cap may reduce the risk of plaque rupture by stabilizing the fibrous cap with extracellular matrix.
Summary and Future Directions
Recent studies suggest that TF plays a nonhemostatic role in many biological processes, and at least some of these effects are mediated via TF–VIIa-induced cell signaling. It is now established that TF–VIIa induces cell signaling via activation of PAR2 and that the ternary complex of TF–VIIa–Xa is a more efficient signaling mediator than the binary TF–VIIa complex, particularly at low concentrations of FVIIa. However, it is currently unclear whether the ternary and the binary complexes activate the same signaling pathways or if each has its own specificities. Because TF–VIIa complex primarily activates PAR2 while TF–VIIa–Xa activates both PAR1 and PAR2, it is expected that there would be some distinction between the signaling mediated by these complexes. Identification of such differences would further reinforce the importance of TF–VIIa signaling. In addition to PARs, other cell surface components, such as integrins, proteoglycans, and growth factor receptors, may also be involved in transmitting or regulating TF–VIIa signaling. Analysis of other potential cell surface receptors that may mediate TF–VIIa signaling is an important area requiring further study. The ability of FVIIa to induce cell signaling is dependent not only on the availability of PARs and TF but also on their spatial proximity. A spatial disconnection between TF and PAR2 may explain why FVIIa fails to induce signaling in some cells, even if they express both TF and PAR2. Currently, there is no information on how the spatial organization of participating signaling components regulates TF–VIIa signaling. This is also another important area in need of further investigation. Additionally, analysis of if and how phosphorylation of the TF cytoplasmic domain regulates TF–VIIa protease-induced signaling also requires future attention. Until now, the TF–VIIa-induced cell signaling studies are primarily limited to in vitro cell model systems and there is no convincing evidence that TF–VIIa signaling actually plays a role in vivo. Thus, the main challenge for future investigators is to establish the importance of TF–VIIa-induced cell signaling in pathophysiological processes in which multiple protease-induced signaling pathways operate simultaneously and redundantly in PAR-mediated signaling. The development of specific inhibitors that suppress TF–VIIa signaling but not TF–VIIa coagulant function or vice versa will provide unique opportunities to design specific drugs that may have therapeutic value in the treatment of diseases that are often associated with aberrant expression of TF.
Acknowledgments
The authors are thankful to Drs Lars Petersen, Brit Sorensen, and Mirella Ezban at Novo-Nordisk, Denmark, for their collaboration on the work related to TF–VIIa signaling. The authors’ work related to this article was supported by National Institutes of Health grants HL65550 and HL58869, and grants from American Heart Association (National and Texas affiliate).
Received September 28, 2004; accepted November 12, 2004.
References
Fleck RA, Rao LVM, Rapaport SI, Varki N. Localization of human tissue factor antigen by immunostaining with monospecific, polyclonal anti-human tissue factor antibody. Thromb Res. 1990; 59: 421–437.
Drake TA, Morrissey JH, Edgington TS. Selective cellular expression of tissue factor in human tissues: Implications for disorders of hemostasis and thrombosis. Am J Pathol. 1989; 134: 1087–1097.
Osterud B. Tissue factor expression by monocytes: regulation and pathophysiological role. Blood Coag Fibrinol. 1998; 9: S9–S14.
Contrino J, Hair G, Kreutzer DL, Rickles FR. In situ detection of tissue factor in vascular endothelial cells: Correlation with the malignant phenotype of human breast disease. Nat Med. 1996; 2: 209–215.
Luther T, Flossel C, Albrecht S, Kotzsch M, Muller M. Tissue factor expression in normal and abnormal mammary gland. Nat Med. 1996; 2: 491–492.
McNamara CA, Sarenbock IJ, Gimple LW, Fenton II JW, Coughlin SR, Owens GK. Thrombin stimulates proliferation of cultured rat aortic smooth muscle cells by a proteolytically activated receptor. J Clin Invest. 1993; 91: 94–98.
Gasic GP, Arenas CP, Gasic TB, Gasic GJ. Coagulation factors X, Xa, and protein S as potent mitogens of cultured aortic smooth muscle cells. Proc Natl Acad Sci U S A. 1992; 89: 2317–2320.
Coughlin SR. Protease-activated receptors in vascular biology. Thromb Haemost. 2001; 86: 298–307.
Pendurthi UR, Rao LVM. Factor VIIa/tissue factor-induced signaling: A link between clotting and disease. In: Litwick G, ed. Vitamins and Hormones. San Diego: Academic Press; 2002: 323–355.
Ruf W, Mueller BM. Tissue factor in cancer angiogenesis and metastasis. Curr Opin Hematol. 1996; 3: 379–384.
Mackman N. Role of tissue factor in hemostasis, thrombosis, and vascular development. Arterioscler Thromb Vasc Biol. 2004; 24: 1015–1022.
Riewald M, Kravchenko VV, Petrovan R, O’brien PJ, Brass LF, Ulevitch RJ, Ruf W. Gene induction by coagulation factor Xa is mediated by activation of PAR-1. Blood. 2001; 97: 3109–3116.
Riewald M, Petrovan RJ, Donner A, Ruf W. Activated protein C signals through the thrombin receptor PAR1 in endothelial cells. J Endotoxin Res. 2003; 9: 317–321.
Pendurthi UR, Ngyuen M, Andrade-Gordon P, Petersen LC, Rao LVM. Plasmin induces Cyr61 gene expression in fibroblasts via protease activated receptor-1 and p44/42 mitogen-activated protein kinase-dependent signaling pathway. Arterioscler Thromb Vasc Biol. 2002; 22: 1421–1426.
Camerer E, Huang W, Coughlin SR. Tissue factor- and factor X-dependent activation of protease-activated receptor-2 by factor VIIa. Proc Natl Acad Sci U S A. 2000; 97: 5255–5260.
Bono F, Schaeffer P, Herault J-P, Michaux C, Nestor A-L, Guillemot J-C, Herbert JM. Factor Xa activates endothelial cells by a receptor cascade between EPR-1 and PAR-2. Arterioscler Thromb Vasc Biol. 2000; 20: e107–e112.
Coughlin SR. Thrombin signalling and protease-activated receptors. Nature. 2000; 407: 258–264.
Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, Plevin R. Proteinase-activated receptors. Pharmacol Rev. 2001; 53: 245–282.
O’brien PJ, Molino M, Kahn M, Brass LF. Protease activated receptors: theme and variations. Oncogene. 2001; 20: 1570–1581.
Bazan JF. Structural design and molecular evolution of a cytokine receptor super family. Proc Natl Acad Sci U S A. 1990; 87: 6934–6938.
Rottingen JA, Enden T, Camerer E, Iversen JG, Prydz H. Binding of human factor VIIa to tissue factor induces cytosolic Ca2+ signals in J82 cells, transfected COS-1 cells, Madin-Darby canine kidney cells and in human endothelial cells induced to synthesize tissue factor. J Biol Chem. 1995; 270: 4650–4660.
Camerer E, Rottingen JA, Iversen JG, Prydz H. Coagulation factors VII and X induce Ca2+ oscillations in Madin-Darby canine kidney cells. J Biol Chem. 1996; 271: 29034–29042.
Prydz HS, Camerer E, Rottingen J-A, Wiiger MT, Gjernes E. Cellular consequences of the initiation of blood coagulation. Thromb Haemost. 1999; 82: 183–192.
Camerer E, Rottingen J-A, Gjernes E, Larsen K, Skartlien AH, Iversen J-G, Prydz H. Coagulation factors VIIa and Xa induce cell signaling leading to up-regulation of the egr-1 gene. J Biol Chem. 1999; 274: 32225–32233.
Poulsen LK, Jacobsen N, Sorensen BB, Bergenhem NCH, Kelly JD, Foster DC, Thastrup O, Ezban M, Petersen LC. Signal transduction via the mitogen-activated protein kinase pathway induced by binding of coagulation factor VIIa to tissue factor. J Biol Chem. 1998; 273: 6228–6232.
Sorensen BB, Freskgard P-O, Nielsen LS, Rao LVM, Ezban M, Petersen LC. Factor VIIa-induced p44/42 mitogen-activated kinase activation requires the proteolytic activity of factor VIIa and is independent of the tissue factor cytoplasmic domain. J Biol Chem. 1999; 274: 21349–21354.
Pendurthi U, Sorensen BB, Petersen LC, Ezban M, Hagel G, Thastrup O, Rao LVM. Factor VIIa induced signaling in fibroblasts. Blood. 2000; 96: 635a.
Camerer E, Gjernes E, Wiiger M, Pringle S, Prydz H. Binding of factor VIIa to tissue factor on keratinocytes induces gene expression. J Biol Chem. 2000; 275: 6580–6585.
Versteeg HH, Hoedemaeker I, Diks SH, Stam JC, Spaargaren M, Bergen en Henegouwen PMP, Deventer SJH, Peppelenbosch MP. Factor VIIa/Tissue factor-induced signaling via activation of Src-like Kinase, Phosphatidylinositol 3-Kinase, and Rac. J Biol Chem. 2000; 275: 28750–28756.
Versteeg HH, Bresser HL, Spek CA, Richel DJ, Deventer SJH. Regulation of the p21 RAS-MAP kinase pathway by factor VIIa. J Thromb Haemost. 2003; 1: 1012–1018.
Wiiger MT, Prydz H. The epidermal growth factor receptor (EGFR) and proline rich tyrosine kinase 2 (PYK2) are involved in tissue factor dependent factor VIIa signalling in HaCaT cells. Thromb Haemost. 2004; 92: 13–22.
Lev S, Moreno H, Martinez R, Canoll P, Peles E, Musacchio JM, Plowman GD, Rudy B, Schlessinger J. Protein tyrosine kinase PYK2 involved in Ca(2+)-induced regulation of ion channel and MAP kinase functions. Nature. 1995; 376: 737–745.
Versteeg HH, Spek CA, Slofstra SH, Diks SH, Richel DJ, Peppelenbosch MP. FVIIa: TF induces cell survival via G12/G13-dependent Jak/STAT activation and BclXL production. Circ Res. 2004; 94: 1032–1040.
Versteeg HH, Sorensen BB, Slofstra S, Van den Brande JHM, Stam JC, van Bergen en Henegouwen PMP, Richel DJ, Petersen LC, Peppelenbosch MP. VIIa/tissue factor interaction results in a tissue factor cytoplasmic domain-independent activation of protein synthesis, p70, and p90 S6 kinase phosphorylation. J Biol Chem. 2002; 277: 27065–27072.
Ollivier V, Bentolila S, Chabbat J, Hakim J, de Prost D. Tissue factor-dependent vascular endothelial growth factor production by human fibroblasts in response to activated factor VII. Blood. 1998; 91: 2698–2703.
Taniguchi T, Kakkar AK, Tuddenham EGD, Williamson RCN, Lemoine NRL. Enhanced expression of urokinase receptor induced through the tissue factor-factor VIIa pathway in human pancreatic cancer. Cancer Res. 1998; 58: 4461–4467.
Wang X, Gjernes E, Prydz H. Factor VIIa induces tissue factor-dependent up-regulation of interleukin-8 in a human keratinocyte line. J Biol Chem. 2002; 277: 23620–23626.
Hjortoe GM, Petersen LC, Albrektsen T, Sorensen BB, Norby PL, Mandal SK, Pendurthi UR, Rao LV. Tissue factor-factor VIIa-specific up-regulation of IL-8 expression in MDA-MB-231 cells is mediated by PAR-2 and results in increased cell migration. Blood. 2004; 103: 3029–3037.
Pendurthi UR, Allen KE, Ezban M, Rao LVM. Factor VIIa and thrombin induce the expression of Cyr61 and connective tissue growth factor, extracellular matrix signaling proteins that could act as possible downstream mediators in factor VII. tissue factor-induced signal transduction. J Biol Chem. 2000; 275: 14632–14641.
Lau LF, Lam S. The CCN family of angiogenic regulators: The integrin connection. Exp Cell Res. 1999; 248: 44–57.
Petersen LC, Thastrup O, Hagel G, Sorensen BB, Freskgard P-O, Rao LVM, Ezban M. Exclusion of known protease activated receptors in factor VIIa-induced signal transduction. Thromb Haemost. 2000; 83: 571–576.
Riewald M, Ruf W. Mechanistic coupling of protease signaling and initiation of coagulation by tissue factor. Proc Natl Acad Sci U S A. 2001; 98: 7742–7747.
Marutsuka K, Hatakeyama K, Sato Y, Yamashita A, Sumiyoshi A, Asada Y. Protease-activated receptor -2 (PAR2) mediates vascular smooth muscle cell migration induced by tissue factor/factor VIIa complex. Thromb Res. 2002; 107: 271–276.
Ishii K, Hein L, Kobilka B, Coughlin SR. Kinetics of thrombin receptor cleavage on intact cells. Relation to signaling. J Biol Chem. 1993; 268: 9780–9785.
Morrissey JH, Macik BG, Neuenschwander PF, Comp PC. Quantitation of activated factor VII levels in plasma using a tissue factor mutant selectively deficient in promoting factor VII activation. Blood. 1993; 81: 734–744.
Wildgoose P, Nemerson Y, Hansen LL, Nielsen FE, Glazer S, Hedner U. Measurement of basal levels of factor VIIa in hemophilia A and B patients. Blood. 1992; 80: 25–28.
Neuenschwander PF, Fiore MM, Morrissey JH. Factor VII autoactivation proceeds via interaction of distinct protease-cofactor and zymogen-cofactor complexes. J Biol Chem. 1993; 268: 21489–21492.
Yamamoto M, Nakagaki T, Kisiel W. Tissue factor dependent autoactivation of human blood coagulation factor VII. J Biol Chem. 1992; 267: 19089–19094.
Rao LVM, Williams T, Rapaport SI. Studies of the activation of factor VII bound to tissue factor. Blood. 1996; 87: 3738–3748.
Ruf W, Dorfleutner A, Riewald M. Specificity of coagulation factor signaling. J Thromb Haemost. 2003; 1: 1495–1503.
Zioncheck TF, Roy S, Vehar GA. The cytoplasmic domain of tissue factor is phosphorylated by a protein kinase C-dependent mechanism. J Biol Chem. 1992; 267: 3561–3564.
Mody RS, Carson SD. Tissue factor cytoplasmic domain peptide is multiply phosphorylated in vitro. Biochem. 1997; 36: 7869–7875.
Dorfleutner A, Ruf W. Regulation of tissue factor cytoplasmic domain phosphorylation by palmitoylation. Blood. 2003; 102: 3998–4005.
Bromberg ME, Konigsberg WH, Madison JF, Pawashe A, Garen A. Tissue factor promotes melanoma metastasis by a pathway independent of blood coagulation. Proc Natl Acad Sci U S A. 1995; 92: 8205–8209.
Mueller BM, Ruf W. Requirement for binding of catalytically active factor VIIa in tissue factor-dependent experimental metastasis. J Clin Invest. 1998; 101: 1372–1378.
Bromberg ME, Sundaram R, Homer RJ, Garen A, Konigsberg WH. Role of tissue factor in metastasis: Functions of the cytoplasmic and extracellular domains of the molecule. Thromb Haemost. 1999; 82: 88–92.
Abe K, Shoji M, Chen J, Bierhaus A, Danave I, Micko C, Casper K, Dillehay DL, Nawroth P, Rickles FR. Regulation of vascular endothelial growth factor production and angiogenesis by the cytoplasmic tail of tissue factor. Proc Natl Acad Sci U S A. 1999; 96: 8663–8668.
Bromberg ME, Garen A, McNiff J, Konigsberg WH. Overexpression of tissue factor does not promote metastasis by upregulation of VEGF or angiogenesis in melanoma cells. Blood. 1997; 90 (suppl): 293a.
Ahamed J, Ruf W. Protease-activated receptor 2-dependent phosphorylation of the tissue factor cytoplasmic domain. J Biol Chem. 2004; 279: 23038–23044.
Belting M, Dorrell MI, Sandgren S, Aguilar E, Ahamed J, Dorfleutner A, Carmeliet P, Mueller BM, Friedlander M, Ruf W. Regulation of angiogenesis by tissue factor cytoplasmic domain signaling. Nat Med. 2004; 10: 502–509.
Taylor FB, Chang A, Ruf W, Morrissey JH, Hinshaw LB, Catlett R, Blick K, Edgington TS. Lethal E. coli septic shock is prevented by blocking tissue factor with monoclonal antibody. Circ Shock. 1991; 33: 127–134.
Creasey AA, Chang ACK, Feigen L, Wun TC, Taylor FB, Hinshaw LB. Tissue factor pathway inhibitor reduces mortality from E. coli septic shock. J Clin Invest. 1993; 91: 2850–2860.
Taylor FB, Chang ACK, Peer G, Li A, Ezban M, Hedner U. Active site inhibited factor VIIa (DEGR VIIa) attenuates the coagulant and interleukin-6 and -8 but not tumor necrosis factor responses of the baboon to LD 100 E. coli. Blood. 1998; 91: 1609–1615.
Taylor FB, Chang ACK, Peer GT, Mather T, Blick K, Catlett R, Lockhart M, Esmon CT. DEGR-factor Xa blocks disseminated intravascular coagulation initiated by E. Coli without preventing shock or organ damage. Blood. 1991; 78: 364–368.
Randolph MM, White GL, Kosanke SD, Bild G, Carr C, Galluppi G, Hinshaw LB, Taylor FB, Jr. Attenuation of tissue thrombosis and hemorrhage by ala-TFPI does not account for its protection against E. coli–a comparative study of treated and untreated non-surviving baboons challenged with LD100 E. coli. Thromb Haemost. 1998; 79: 1048–1053.
Miller DL, Welty-wolf KE, Carraway MS, Ezban M, Ghio A, Suliman H, Piantadosi CA. Extrinsic coagulation cascade blockade attenuates lung injury and proinflammatory cytokine release after intratracheal lipopolysaccharide. Am J Respir Cell Mol Biol. 2002; 26: 650–658.
Randolph GJ, Luther T, Albrecht S, Magdolen V, Muller WA. Role of tissue factor in adhesion of mononuclear phagocytes to and trafficking through endothelium in vitro. Blood. 1998; 92: 4167–4177.
Cunningham MA, Romas P, Hutchinson P, Holdsworth SR, Tipping PG. Tissue factor and factor VIIa receptor/ligand interactions induce proinflammatory effects in macrophages. Blood. 1999; 94: 3413–3420.
Pawlinski R, Pedersen B, Schabbauer G, Tencati M, Holscher T, Boisvert W, Andrade-Gordon P, Frank RD, Mackman N. Role of tissue factor and protease-activated receptors in a mouse model of endotoxemia. Blood. 2004; 103: 1342–1347.
Sharma L, Melis E, Hickey MJ, Clyne CD, Erlich J, Khachigian LM, Davenport P, Morand E, Carmeliet P, Tipping PG. The cytoplasmic domain of tissue factor contributes to leukocyte recruitment and death in endotoxemia. Am J Pathol. 2004; 165: 331–340.
Browder T, Folkman J, Pirie-Shepherd S. The hemostatic system as a regulator of angiogenesis. J Biol Chem. 2000; 275: 1521–1524.
Zhang Y, Deng Y, Luther T, Muller M, Ziegler R, Waldherr R, Stern DM, Nawroth P. Tissue factor controls the balance of angiogenic and antiangiogenic properties of tumor cells in mice. J Clin Invest. 1994; 94: 1320–1327.
Li A, Dubey S, Varney ML, Dave BJ, Singh RK. IL-8 directly enhanced endothelial cell survival. proliferation, and matrix metalloproteinases production and regulated angiogenesis. J Immunol. 2003; 170: 3369–3376.
Kireeva ML, Lam S, Lau LF. Adhesion of human umbilical vein endothelial cells to the immediate-early gene product cyr61 is mediated through integrin v?3. J Biol Chem. 1998; 273: 3090–3096.
Planque N, Perbal B. A structural approach to the role of CCN (CYR61/CTGF/NOV) proteins in tumourigenesis. Cancer Cell Int. 2003; 3: 15.
Dorfleutner A, Hintermann E, Tarui T, Takada Y, Ruf W. Crosstalk of Integrin {alpha}3{beta}1 and Tissue Factor in Cell Migration. Mol Biol Cell. 2004; 15: 4416–4425.
Ott I, Fischer EG, Miyagi Y, Mueller BM, Ruf W. A role for tissue factor in cell adhesion and migration mediated by interaction with actin-binding protein 280. J Cell Biol. 1998; 140: 1241–1253.
Rao LVM. Tissue factor as a tumor procoagulant. Cancer Metastasis Rev. 1992; 11: 249–266.
Hamada K, Kuratsu J, Saitoh Y, Nishi T, Ushio Y. Expression of tissue factor expression correlates with grade of malignancy in human glioma. Cancer. 1996; 77: 1877–1883.
Kakkar AK, Lemoine NR, Scully MF, Tebbutt S, Williamson RC. Tissue factor expression correlates with histological grade in human pancreatic cancer. Br J Surg. 1995; 82: 1101–1104.
Shigemori C, Wada H, Matsumoto K, Shiku H, Nakamura S, Suzuki H. Tissue factor expression and metastatic potential of colorectal cancer. Thromb Haemost. 1998; 80: 894–898.
Seto S, Onodera H, Kaido T, Yoshikawa A, Ishigami S, Arii S, Imamura M. Tissue factor expression in human colorectal carcinoma: correlation with hepatic metastasis and impact on prognosis. Cancer. 2000; 88: 295–301.
Mueller BM, Reisfeld RA, Edgington TS, Ruf W. Expression of tissue factor by melanoma cells promotes efficient hematogenous metastasis. Proc Natl Acad Sci U S A. 1992; 89: 11832–11836.
Dimmeler S, Zeiher AM. Endothelial cell apoptosis in angiogenesis and vessel regression. Circ Res. 2000; 87: 434–439.
Bergmann A, Agapite J, McCall K, Steller H. The Drosophila gene hid is a direct molecular target of Ras-dependent survival signaling. Cell. 1998; 95: 331–341.
Yu J, Zhang L. Apoptosis in human cancer cells. Curr Opin Oncol. 2004; 16: 19–24.
Debatin KM. Apoptosis pathways in cancer and cancer therapy. Cancer Immunol Immunother. 2004; 53: 153–159.
Versteeg HH, Spek CA, Richel DJ, Peppelenbosch MP. Coagulation factors VIIa and Xa inhibit apoptosis and anoikis. Oncog. 2004; 23: 410–417.
Sorensen BB, Rao LVM, Tornehave D, Gammeltoft S, Petersen LC. Antiapoptotic effect of coagulation factor VIIa. Blood. 2003; 102: 1708–1715.
D’Andrea MR, Derian CK, Santulli RJ, Andrade-Gordon P. Differential expression of protease-activated receptors-1 and -2 in stromal fibroblasts of normal, benign, and malignant human tissues. Am J Pathol. 2001; 158: 2031–2041.
Even-ram SC, Uziely B, Cohen P, Grisaru-granovsky S, Reich R, Bar-Shavit R, Giocondi M. Thrombin receptor overexpression in malignant and physiological invasion processes. Nat Med. 1998; 4: 909–914.
Fischer EG, Ruf W, Mueller BM. Tissue factor-initiated thrombin generation activates the signalling thrombin receptor on malignant melanoma cells. Cancer Res. 1995; 55: 1629–1632.
Tellez C, Bar-Eli M. Role and regulation of the thrombin receptor (PAR-1) in human melanoma. Oncogene. 2003; 22: 3130–3137.
Nierodzik ML, Chen K, Takeshita K, Li JJ, Huang YQ, Feng XS, D’Andrea MR, Andrade-Gordon P, Karpatkin S. Protease-activated receptor 1 (PAR-1) is required and rate-limiting for thrombin-enhanced experimental pulmonary metastasis. Blood. 1998; 92: 3694–3700.
Bromberg ME, Bailly MA, Konigsberg WH. Role of protease-activated receptor 1 in tumor metastasis promoted by tissue factor. Thromb Haemost. 2001; 86: 1210–1214.
Even-ram SC, Maoz M, Pokroy E, Reich R, Katz B-Z, Gutwein P, Altevogt P, Bar-Shavit R. Tumor cell invasion is promoted by activation of protease activated receptor-1 in cooperation with the v?5 integrin. J Biol Chem. 2001; 276: 10952–10956.
Darmoul D, Gratio V, Devaud H, Lehy T, Laburthe M. Aberrant expression and activation of the thrombin receptor protease-activated receptor-1 induces cell proliferation and motility in human colon cancer cells. Am J Pathol. 2003; 162: 1503–1513.
Shi X, Gangadharan B, Brass LF, Ruf W, Mueller BM. Protease-activated receptors (PAR1 and PAR2) contribute to tumor cell motility and metastasis. Mol Cancer Res. 2004; 2: 395–402.
Camerer E, Qazi AA, Duong DN, Cornelissen I, Advincula R, Coughlin SR. Platelets, protease-activated receptors, and fibrinogen in hematogenous metastasis. Blood. 2004; 104: 397–401.
Werner S, Grose R. Regulation of wound healing by growth factors and cytokines. Physiol Rev. 2003; 83: 835–870.
Martin P. Wound healing–aiming for perfect skin regeneration. Science. 1997; 276: 75–81.
Chen C-C, Mo F-E, Lau LF. The angiogenic factor Cyr61 activates a genetic program for wound healing in human skin fibroblasts. J Biol Chem. 2001; 276: 47329–47334.
Wang JF, Olson ME, Ball DK, Brigstock DR, Hart DA. Recombinant connective tissue growth factor modulates porcine skin fibroblast gene expression. Wound Repair Regen. 2003; 11: 220–229.
Ardissino D, Merlini PA, Arlens R, Coppola R, Bramucci E, Lucreziotti S, Repetto A, Fetiveau R, Mannucci PM. Tissue factor in human coronary atherosclerotic plaques. Clinica Chimica Acta. 2000; 291: 235–240.
Ardissino D, Merlini PA, Bauer KA, Bramucci E, Ferrario M, Coppola R, Fetiveau R, Lucreziotti S, Rosenberg RD, Mannucci PM. Thrombogenic potential of human coronary atherosclerotic plaques. Blood. 2001; 98: 2726–2729.
Marmur JD, Thiruvikraman SV, Fyfe BS, Guha A, Sharma SK, Ambrose JA, Fallon JT, Nemerson Y, Taubman MB. Identification of active tissue factor in human coronary atheroma. Circulation. 1996; 94: 1226–1232.
Taubman MB, Fallon JT, Schecter AD, Giesen P, Mendlowitz M, Fyfe BS, Marmur JD, Nemerson Y. Tissue factor in the pathogenesis of atherosclerosis. Thromb Haemost. 1997; 78: 200–204.
Sato Y, Asada Y, Marutsuka K, Hatakeyama K. Tissue factor induces migration of cultured aortic smooth muscle cells. Thromb Haemost. 1996; 75: 389–392.
Sato Y, Kataoka H, Asada Y, Marutsuka K, Kamikubo YI, Koono M, Sumiyoshi A. Overexprssion of tissue factor pathway inhibitor in aortic smooth muscle cells inhibits cell migration induced by tissue factor/factor VIIa complex. Thrombosis Res. 1999; 94: 401–406.
Singh R, Pan S, Mueske CS, Witt T, Kleppe LS, Peterson TE, Slobodova A, Chang J-Y, Caplice NM, Simari RD. Role for tissue factor pathway in murine model of vascular remodeling. Circ Res. 2001; 89: 71–76.
Siegbahn A, Johnell M, Rorsman C, Ezban M, Heldin C-H, Ronnstrand L. Binding of factor VIIa to tissue factor on human fibroblasts leads to activation of phospholipase C and enhanced PDGF-BB-stimulated chemotaxis. Blood. 2000; 96: 3452–3458.
Cirillo P, Cali G, Golino P, Calabro P, Forte L, De Rosa S, Pacileo M, Ragni M, Scopacasa F, Nitsch L, Chiariello M. Tissue factor binding of activated factor VII triggers smooth muscle cell proliferation via extracellular signal-regulated kinase activation. Circulation. 2004; 109: 2911–2916.
Mandal SK, Rao LVM, Tran TT, Pendurthi UR. A novel mechanism of plasmin-induced mitogenesis in fibroblasts. J Thromb Haemost 2004. In press.
Herbert JM, de Prost D, Ollivier V, Melis E, Carmeliet P. Tissue factor is not involved in the mitogenic activity of factor VIIa. Biochem Biophys Res Commun. 2001; 281: 1074–1077.
Oemar BS, Werner A, Garnier JM, Do DD, Godoy N, Nauck M, Marz W, Rupp J, Pech M, Luscher TF. Human connective tissue growth factor is expressed in advanced atherosclerotic lesions. Circulation. 1997; 95: 831–839.
Oemar BS, Luscher TF. Connective tissue growth factor: Friend or foe? Arterioscler Thromb Vasc Biol. 1997; 17: 1483–1489.
Schober JM, Chen N, Grzeszkiewicz TM, Jovanovic I, Emeson EE, Ugarova TP, Ye RD, Lau LF, Lam SC. Identification of integrin alpha(M)beta(2) as an adhesion receptor on peripheral blood monocytes for Cyr61 (CCN1) and connective tissue growth factor (CCN2): immediate-early gene products expressed in atherosclerotic lesions. Blood. 2002; 99: 4457–4465.
Hilfiker A, Hilfiker-Kleiner D, Fuchs M, Kaminski K, Lichtenberg A, Rothkotter HJ, Schieffer B, Drexler H. Expression of CYR61, an angiogenic immediate early gene, in arteriosclerosis and its regulation by angiotensin II. Circulation. 2002; 106: 254–260.
Wilcox JN, Noguchi S, Casanova J. Extrahepatic synthesis of factor VII in human atherosclerotic vessels. Arterioscler Thromb Vasc Biol. 2003; 23: 136–141.
Jedsadayanmate A, Chen C-C, Kireeva ML, Lau LF. Activation-dependent adhesion of human platelets to Cyr61 and Fisp12/mouse connective tissue growth factor is mediated through integrin aIIb?3. J Biol Chem. 1999; 274: 24321–24327.(L. Vijaya Mohan Rao; Usha)
Correspondence to L. Vijaya Mohan Rao, PhD, Biomedical Research, The University of Texas Health Center at Tyler, 11937 US Highway 271, Tyler, TX 75708. E-mail vijay.rao@uthct.edu
Abstract
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.
Recent studies show that tissue factor–factor VIIa, whose primary function is to initiate the clotting cascade, transduces cell signaling in various cell types. This brief review summarizes recent literature on potential mechanisms by which tissue factor–factor VIIa activates cell signaling, and how tissue factor–factor VIIa-induced cell signaling may affect various pathophysiological processes.
Key Words: tissue factor ? factor VIIa ? protease activated receptors ? cell signaling
Introduction
The close link between coagulation and various diseases, such as sepsis, atherosclerosis, and tumor metastasis, suggests a complex interplay between the clotting cascade and disease progression. Numerous studies have demonstrated that coagulation proteases can function like hormones to regulate cellular behavior. For example, thrombin, the principal protease generated during coagulation, has been shown to activate platelets and regulate the behavior of other cells by transmitting signals via activation of G protein-coupled protease activated receptors (PARs). Although there has been initial skepticism on whether other clotting proteases can also activate PARs, recent studies provide convincing evidence that many proteases involved in clotting can indeed activate PARs and regulate cellular behaviors at physiological concentrations. One such protease, FVIIa, the physiological initiator of the coagulation cascade, has received much attention lately. The focus of the present article is to review briefly recent developments in tissue factor (TF)–VIIa proteolytic activity-mediated cell signaling.
TF–Factor VIIa
TF is a transmembrane cellular receptor for FVII/FVIIa. Binding of plasma FVII/FVIIa to TF triggers the coagulation cascade, which leads to thrombin generation that subsequently stimulates platelet activation and cleaves fibrinogen. Under normal conditions, TF is constitutively expressed in many cell types, including fibroblasts and pericytes in and surrounding blood vessel walls, but is not expressed in blood cells or the endothelial cells that line blood vessels.1,2 However, under certain pathological conditions, such as sepsis and cancer, monocytes3 and endothelial cells4 express TF, although the latter finding has not been confirmed by others.5 Thus, blood vessel wall injury or certain disease conditions permit FVII/FVIIa interaction with TF on cell surfaces. The activation of TF-induced coagulation pathway not only leads to fibrin formation but also contributes to vascular remodeling, which is caused by growth factors secreted by activated platelets, as well as the intermediary products factor Xa and thrombin that promote vascular smooth cell proliferation6,7 and alter endothelium.8 TF expression has been linked directly to various pathophysiological processes, such as development, inflammation, and tumor metastasis.9–11
PARs
Certain proteases control cellular functions via signaling through specific membrane receptors known as PARs, members of a larger family of 7 transmembrane cell surface receptors that mediate cell activation via G-proteins. At present, there are 4 known PARs: PAR1, PAR2, PAR3, and PAR4. PAR1, the prototypic family member, is cleaved primarily by thrombin, but can also be cleaved by other proteases, such as FXa,12 activated protein C (APC),13 plasmin,14 and FVIIa.15 PAR3 and PAR4 are also cleaved by thrombin, whereas PAR2 is cleaved by the trypsin-like enzymes, FXa16 and FVIIa.15 Although the mechanism of PARs activation was initially established for thrombin and PAR1, the mechanism is similar for the other PARs. In short, the protease cleaves the receptor at a specific site in the amino-terminal extracellular domain, leading to the unmasking of a new N-terminus. The new N-terminus then acts as a tethered ligand, binding intramolecularly to the body of the receptor to initiate transmembrane signaling. In fact, synthetic peptides mimicking the tethered ligand can activate the receptor independent of its cleavage. A number of excellent reviews provide details of the mechanism of activation of PARs and define the role for these receptors in vivo.17–19
TF–VIIa-Induced Signaling
Because aberrant expression of TF is associated with various diseases and TF is structurally similar to members of the class 2 cytokine receptor family,20 there has been a great interest in examining the role of TF in cell signaling. The first evidence for TF–VIIa-induced signaling came from studies in which FVIIa binding to TF was shown to induce intracellular Ca2+ oscillations in a number of TF-expressing cells.21,22 This FVIIa-induced calcium signaling required the binding of catalytically active FVIIa to TF but did not require the presence of TF cytoplasmic domain. Although cells that express TF, either inducibly or constitutively, responded to FVIIa binding, the fraction of cells that released Ca2+ and the extent to which Ca2+ was released varies among different cell types.23 Interestingly, some cell lines, such as HK-2 (a kidney cell line that constitutively expresses TF) and CHO cells stably transfected with TF, failed to respond to FVIIa, despite abundant expression of TF on their surfaces.24
Work from other laboratories demonstrated that FVIIa interaction with TF on a variety of cells, including fibroblasts, epithelial cells, and endothelial cells, activated multiple signaling pathways. Poulsen et al25 reported first that FVIIa binding to BHK cells that are stably transfected with human TF [BHK(TF)] resulted in a transient activation of p44/42 MAPK. Further studies showed that the FVIIa-induced p44/42 MAPK activation is dependent on proteolytically active FVIIa, but independent of the TF cytoplasmic domain.26 FVIIa is shown to activate p44/42 MAPK in other cell types that express TF, such as fibroblasts27 and keratinocytes,28 but the response is not as robust as that observed in BHK(TF) cells. Additionally, FVIIa treatment of keratinocytes also increased the phosphorylation of key components of the other 2 MAPK pathways, p38 and C-Jun N-terminal kinase (JNK).24 Versteeg et al29 demonstrated that FVIIa stimulates a signaling pathway in fibroblasts (A14 cells), leading to the activation of the Src-like family members c-Src, Lyn, and Yes, and subsequently PI3-kinase, which then induces the stimulation of p44/42 MAPK, c-Akt/protein kinase B, and the small GTPases Rac and Cdc42. This group also showed that FVIIa-induced p44/42 MAPK activation is mediated via p21 Ras activation in BHK(TF) and HaCaT keratinocytes.30 In HaCaT cells, FVIIa has been shown to phosphorylate PYK2,31 a kinase that has been implicated in the regulation of MAP kinase activation.32 In addition to activating the p21 ras/MAPK pathway, FVIIa has also been shown to induce STAT5 phosphorylation via Jak2 activation in BHK(TF) cells33 and to stimulate the protein synthesis machinery via activation of p70/p85s6K, p90RSK, and eventually eukaryotic initiation factor eIF-4E.34 Although the described data provide convincing evidence that TF–VIIa activates multiple signaling pathways that could affect various cellular processes, one should exercise caution in extrapolating these data because the majority of these data were derived from a single cell line, BHK(TF).
To address how TF-mediated cell signaling could potentially contribute to various pathophysiological conditions, several groups have focused on investigating TF–VIIa-induced alterations in gene expression. Examination of specific gene transcripts, whose products were believed to be pathophysiologically relevant to diseases associated with aberrant expression of TF, revealed that exposure of TF-expressing cells (fibroblasts, tumor cells, or keratinocytes) to FVIIa led to increased expression of vascular endothelial growth factor (VEGF),35 uPAR,36 Egr-1,24 and IL-8.37,38 Global analysis of TF–VIIa protease-induced signaling on the transcriptional machinery revealed that FVIIa binding to TF on fibroblasts or keratinocytes alters the expression of a few select genes. One of the upregulated transcripts observed in differential display polymerase chain reaction was identified as a poly(A) polymerase, whose product plays an important role in the processing of mRNA. Microarray analysis of fibroblasts briefly exposed to FVIIa revealed that FVIIa upregulates the expression of Cyr61 (CCN1) and connective tissue growth factor (CCN2).39 CCN1 and CCN2 are extracellular matrix signaling proteins that were recently shown to regulate a myriad of cellular functions, such as cell adhesion, proliferation, migration, and tumor metastasis.40 Using low-density cDNA arrays, Camerer et al28 showed that FVIIa interaction with TF on keratinocytes upregulates several genes that are relevant to the wound repair process.
TF–VIIa Activation of a PAR
Because TF–VIIa-induced signaling requires catalytically active FVIIa and is independent of the TF cytoplasmic tail, it has been hypothesized that TF–VIIa transmits cell signaling via PAR activation. However, until recently there was confusion about whether TF–VIIa mediated the cell signaling via 1 of the 4 known PARs or involved the activation of a novel PAR. The lack of heterologous desensitization (ie, failure of PAR agonists to abolish the response induced by subsequently added VIIa or vice versa), the differences between PAR agonists and FVIIa for their ability to induce Ca2+ release and p44/42 MAPK activation, and the lack of FVIIa-induced response in cells that express TF and the known PARs indicated that FVIIa may induce intracellular signaling via activation of a novel PAR or requires, in addition to a known PAR, an additional cell surface component.21,24,26,39,41 However, a recent study by Camerer et al15 showed that FVIIa induces Ca2+ release in Xenopus oocytes transfected with TF together with PAR1 or PAR2, but not PAR3 and PAR4. Similarly, transfection of lung fibroblasts from PAR1-deficient mice with TF and PAR2 conferred the FVIIa-induced response.15 These data suggest that TF–VIIa activates PAR2 and, to a lesser degree, PAR1. This conclusion is further supported by the observation that FVIIa can induce p44/42 MAPK activation in CHO cells transfected with TF and PAR2 but not in CHO cells expressing TF alone.42 Additionally, recent data38 demonstrate that specific antibodies against PAR2 but not PAR1 block FVIIa-induced IL-8 gene expression and cell migration in breast carcinoma cells. Similarly, PAR2 antibodies were shown to block TF–VIIa-induced smooth muscle cell migration.43 Together, these data suggest that TF–VIIa transmits cell signaling via activation of PAR2 (Figure 1). However, this does not mean that TF–VIIa cannot transmit signals via receptors other than PAR2.
Figure 1. A schematic representation of TF–VIIa protease-induced signaling. TF–VIIa activates PAR2 and the ternary complex of TF–VIIa–Xa activates both PAR1 and PAR2. Activation of PAR2-specific signaling pathway may lead to phosphorylation of TF cytoplasmic tail. Phosphorylation of TF cytoplasmic domain releases its negative regulatory control of PAR2-mediated signaling.
At present, it is unclear why FVIIa fails to induce calcium signaling in a number of cell types, including fibroblasts, epithelial cells, and tumor cells that express abundant TF and one or more PARs (PAR1 and PAR2)27,38,41 (and also unpublished data of the authors), and respond robustly to FVIIa stimulation by activating p44/42 MAPK25,41 or gene expression.39 In fact, failure to induce Ca2+ release and the lack of the desensitizing effect of FVIIa on PAR1 and PAR2 agonist-induced Ca2+ release lead to a conclusion, which in retrospective is not well-justified, that TF–VIIa-induced intracellular signaling is not mediated via known PARs but may involve proteolytic cleavage of an unidentified member of the PAR family.41 Why does activation of PAR2/PAR1 by TF–VIIa, in contrast to their activation by thrombin, trypsin, or PAR agonist peptides, fail to induce Ca2+ mobilization? One possible explanation may be an inefficient, but physiologically important, cleavage of PAR2/PAR1 by TF–VIIa. Earlier studies44 have shown that the magnitude of the PAR1-mediated protease response is determined by both the rate and extent of receptor cleavage. A low concentration of protease or in the presence of an inefficient protease, only a limited number of receptors would be activated. Although a limited number of activated receptors could transduce a signal, a measurable response requires activation of a minimum number of receptors at a certain rate.19 Additionally, the rate and extent of receptor activation required to elicit a specific response may be dependent on the response that is measured. For example, when receptor cleavage correlates with phosphoinositide hydrolysis, IP3 formation is proportional to the absolute amount of cleaved receptor, but the subsequent increase in cytosolic Ca2+ occurs only if IP3 is generated quickly enough to accumulate.44 However, this reasoning may not fully explain the phenomenon described given that FVIIa (10 nM), trypsin (1 nM), and PAR2 peptide agonist (1 μmol/L) treatments all result in a similar rate of IP3 hydrolysis, yet only trypsin and PAR2 peptide agonist, and not FVIIa, produced a clear increase in Ca2+ release (unpublished data of authors).
Although the TF–VIIa complex is sufficient to induce cell signaling, it is unclear whether this binary complex functions as an efficient signaling unit in vivo. In many of the studies described here, high concentrations (10 to 100 nM) of FVIIa were required to obtain a measurable signaling response.15,21,26,42 It is likely that such high concentrations of FVIIa are needed to saturate TF rapidly, which may be essential for measuring the signaling response using a short-term assay, such as Ca2+ release or MAPK activation. In fact, low concentrations of FVIIa (5 to 10 nM) are shown to be capable of producing a pronounced response when signaling is analyzed using a long-term assay, such as gene expression28,38,39 However, in circulation most of the FVII is in zymogen form. FVIIa concentration in the plasma is 1% or less (100 pM) of the total circulating FVII (10 nM).45,46 Thus, it is crucial to show that traces of FVIIa in FVII are sufficient to induce cell signaling. However, it is pertinent to note here that because FVII bound to TF can be autocatalytically converted to FVIIa,47–49 most of the FVII bound to cell surface TF will be converted to FVIIa. Consistent with this notion, we found that plasma concentration of zymogen FVII added to fibroblasts39 and tumor cells38 induced Cyr61 and IL-8 gene expression, respectively, with a slight delay. The delay probably reflects the time required for autoactivation of FVII. When substrate FX is present at plasma concentrations, picomolar concentrations of FVIIa elicit a robust signal in cells expressing TF and PAR2.15 This suggests that the TF–VIIa-generated FXa is capable of signaling independent of thrombin. Riewald and Ruf found that the activation of FX by the TF–VIIa complex resulted in a much more robust signal than that induced by the TF–VIIa complex alone, free FXa, or FXa that was generated in situ by the intrinsic activation complex.42 These data, coupled with additional studies using a unique inhibitor (NAPc2, nematode anticoagulant protein C2) that preserves FXa activity in the complex while inhibiting free FXa and TF–VIIa proteolytic activity, revealed that the transient ternary TF–VIIa–Xa complex is a potent signaling unit in which FXa efficiently activates both PAR1 and PAR2.42 This finding supports the hypothesis that upstream coagulation protease signaling is mechanistically coupled to the initiation of the coagulation pathway. Although our data support the concept that the ternary complex of TF–VIIa–Xa is a more potent activator of PAR2 than the binary TF–VIIa complex, this difference is abolished when TF sites are saturated with FVIIa (Figure 2).38 It is currently difficult to conclude whether the ternary or the binary complex is the primary signaling unit in vivo. It is likely that both complexes play a role in vivo, and which complex is more active may depend on multiple factors, such as the levels of TF and PAR2/PAR1 expression, the availability of FX, and the localization/organization of these components on the cell membrane.
Figure 2. A comparison of efficiency of TF–VIIa-mediated and TF–VIIa–Xa-mediated cell signaling. MDA-MB-231 cells were stimulated with varying concentrations of FVIIa in the presence or absence of substrate FX (175 nM). The signaling was evaluated in either IP3 hydrolysis assay (A) or IL-8 gene expression (B).
Although it is apparent that TF–VIIa elicits cell signaling via activation of PAR2, it is unclear whether other cell membrane components participate in the signaling process. It is interesting to note that in some cases, even a high concentration of FVIIa fails to elicit a signaling response, even in cells expressing both functional TF and PAR2.24,50 It raises a possibility that other cell components may contribute or regulate TF–VIIa-induced cell signaling. In this context, it is important to note that Wiiger and Prydz31 recently demonstrated that the epidermal growth factor receptor is involved in the transduction of the TF–VIIa signal in HaCaT cells. However, the role of epidermal growth factor receptor in TF–VIIa-induced signaling still needs to be confirmed.
TF Cytoplasmic Domain-Dependent Signaling
In heterologous expression studies, the TF cytoplasmic domain is not clearly required for the TF–VIIa-induced cell signaling.24,26,34 However, it is unclear whether TF, as a true receptor, can transmit cell signaling via its cytoplasmic tail, or whether the cytoplasmic domain can regulate the protease-induced signaling. Analysis of human TF protein sequence revealed a consensus sequence for protein kinase C phosphorylation in the cytoplasmic domain, which is shown to be phosphorylated in response to phorbol esters.51 Mody and Carson52 showed, using a synthetic peptide corresponding to residues 245 to 263 of the human TF cytoplasmic domain and glioblastoma cell extract (as a source of kinase activity), that TF cytoplasmic domain is phosphorylated at multiple serine residues. Recent studies show that protein kinase C-dependent phosphorylation of Ser253 enhances subsequent Ser258 phosphorylation by a proline-directed kinase.53 A functional role for the TF cytoplasmic domain was documented in hematogenous metastasis. Deletion of the cytoplasmic domain54,55 or mutation of the cytoplasmic phosphorylation sites Ser253 and Ser25855,56 are shown to reduce the TF-induced metastasis. Abe et al57 showed that the TF cytoplasmic domain, independent of FVIIa, is responsible for the upregulation of VEGF in melanoma cells transfected with TF. However, others failed to confirm this finding.58 Recent studies with transgenic mice that lack the cytoplasmic domain of TF (but have normal coagulant function) indicate that the cytoplasmic domain of TF contributes to NF-B activation, pro-inflammatory cytokine production, and leukocyte recruitment after endotoxin challenge. However, further studies are needed to confirm that the TF cytoplasmic domain is directly responsible for the observed effects. Other studies suggest that the TF cytoplasmic domain contributes to cell signaling indirectly, ie, by modulating TF–VIIa-induced cell signaling. For example, deletion of the TF cytoplasmic domain is shown to impair TF–VIIa protease activity-induced reactive oxygen species production in monocytes. Elegant studies performed recently by Ahamed et al show that TF–VIIa–Xa activation of PAR2 induces TF cytoplasmic domain phosphorylation,59 and this phosphorylation of the TF cytoplasmic domain releases its negative regulatory control of PAR2 signaling-mediated angiogenesis60 (Figure 2). At present, it is unclear whether this regulatory mechanism is specific for endothelial cells and the angiogenic process or if it also plays a role in modulating other PAR2 signaling-mediated events in other cell types.
A Role for TF–VIIa Signaling in Pathophysiology
It is widely accepted that TF, in addition to its role in coagulation, may contribute to various pathophysiological processes. However, it is unclear whether TF–VIIa-induced (or TF–VIIa–Xa-induced) cell signaling contributes directly to these biological processes, or whether cell signaling induced by coagulation proteases generated by TF–VIIa, such as thrombin in concert with an end product fibrin, fully accounts for the altered cellular processes. Recent studies suggest that both mechanisms are involved. The following discussion is limited to the potential role of direct TF–VIIa-induced cell signaling in a select few biological processes (Figure 3). For discussion on the role of TF in development, the reader is referred to a recent review.11
Figure 3. TF–VIIa signaling mediated cellular effects and their contribution to various pathophysiological processes (see text for details).
Inflammation
It is well-established that inflammatory mediators activate coagulation by inducing TF expression on blood mononuclear cells and probably on vascular endothelium. In turn, expression of TF on these cells appears to regulate the inflammatory response. The observation that during sepsis the TF-initiated coagulation pathway induces a lethal inflammatory escalation even when fibrin formation and microthrombosis are effectively blocked via decreased thrombin generation61–65 suggests that TF–VIIa cell signaling induces a pro-inflammatory response. Additionally, inhibition of the TF–VIIa complex during septicemia significantly reduces the inflammatory response, as measured by decreased IL-6 and IL-8 plasma levels and reduced inflammatory changes in the lung, such as neutrophil infiltration and edema.63,66 In vitro studies67 also indicate that TF plays an important role in reverse transmigration of mononuclear phagocytes, a process that occurs during the resolution of acute inflammation. FVIIa binding to TF has been shown to augment the macrophage pro-inflammatory functions, such as the production of reactive oxygen species and the expression of major histocompatability complex class II and cell adhesion molecules, both in vivo and in vitro.68 Furthermore, recent studies using the mouse model of endotoxemia have shown that genetically modified mice expressing low levels of TF exhibited reduced IL-6 induction and increased survival compared with control mice.69 Although a deficiency of either PAR1 or PAR2 has no effect on inflammation or survival, a combination of thrombin inhibition and PAR2 deficiency reduces both inflammation and mortality similar to that observed in the low-TF mice.69 These data suggest that PAR1 and PAR2 may have partially redundant roles and contribute to the link between coagulation and inflammation. Together, these data imply that TF–VIIa-induced cell signaling via PAR2 may contribute to pro-inflammation. However, it is difficult to conclude from these results whether the pro-inflammatory signaling unit is TF–VIIa, TF–VIIa–Xa, or FXa, given that all of them are capable of activating PAR2. Furthermore, the reduced lipopolysaccharide-induced IL-6 expression and enhanced survival in the low-TF mice may be unrelated to the defect in TF–VIIa protease-induced signaling given that similar effects were observed in mice expressing TF that lacks the cytoplasmic domain and has normal coagulant function.70 However, one should exercise caution in interpreting the data from the latter studies because these studies were performed in mice with a mixed genetic background, which likely leads to a high degree of variability.
Tumor Angiogenesis
Hemostasis and angiogenesis are interrelated processes. Proteins generated by the hemostatic system are known to have regulatory effects on angiogenesis.71 TF-induced coagulation can indirectly support angiogenesis in several ways, such as the release of positive and negative regulators from activated platelets, thrombin signaling via endothelial cell PAR1, and the generation of provisional fibrin-rich matrix. Whether TF–VIIa signaling plays a direct role in the regulation of angiogenesis is not entirely clear. Overexpression of TF in tumor cells leads to the upregulation of the pro-angiogenic factor VEGF and the downregulation of the anti-angiogenic protein thrombospondin-1.72 However, Bromberg et al found no correlation between TF expression and VEGF.56 Recent studies have shown that TF–VIIa induces IL-8 production via PAR2 signaling, and the secreted IL-8 is capable of stimulating tumor cell migration and invasion in an autocrine fashion.38 IL-8 secreted by tumor cells was shown previously to regulate angiogenesis directly by enhancing endothelial cell survival, proliferation, and matrix metalloproteinase production.73 Surprisingly, although many cancer cell lines express functional PAR1, PAR2, and TF, these cells respond to either thrombin or FVIIa, but not to both in increasing IL-8 (unpublished data of authors). This observation suggests that other cell components, either on the cell surface or within the cell, provide further specificity for the protease-induced signaling.
In cancer cells (unpublished data of the authors) and fibroblasts,39 TF–VIIa signaling upregulates the expression of CCN1, a novel matrix signaling protein that is a ligand to integrin v?3 on endothelium.74 Integrin v?3, an adhesion receptor known to be involved in signaling, regulates a number of cellular processes, including angiogenesis and tumor metastasis. The stimulatory effects of CCN1 on cell proliferation, migration, and survival via its interaction with various integrins are thought to be responsible for its role in angiogenesis and tumorigenesis.75 In addition to CCN1 and IL-8, TF–VIIa-induced cell signaling may also upregulate a number of other gene products, including uPAR,36 which plays a regulatory role in angiogenesis. Microarray analyses of MDA-MB-231 breast carcinoma cells exposed to FVIIa and a control vehicle show that FVIIa induces a set of genes whose products play a role in various steps of angiogenesis and tumor growth. The gene products include chemokines, cytokines, growth factors, cell adhesion proteins, and proteins involved in cell cycle control, apoptosis, and inflammation (unpublished data). Together, these data support the hypothesis that TF–VIIa cell signaling may play an important role in angiogenesis regulation (Figure 4).
Figure 4. TF–VIIa signaling and angiogenesis. Binding of FVIIa to tumor cell TF activates a genetic program, which leads to upregulation of various chemokines, cytokines, and growth factors, including IL-8, CCN1, and VEGF. These secreted cytokines and growth factors from tumor cells promote endothelial cell proliferation and migration, leading to angiogenesis.
Recent studies indicate a complex role for TF in angiogenesis. Belting et al60 show that genetic deletion of the TF cytoplasmic domain enhances PAR2-dependent angiogenesis, presumably in synergy with platelet-derived growth factor BB. In neonatal mice, the diameter of the superficial vascular plexus of TF–cytoplasmic domain-deleted mice was twice that observed in wild-type mice, indicating that the TF cytoplasmic tail negatively regulates in vivo angiogenesis during postnatal development. Furthermore, ocular tissues from diabetic patients display a colocalization of PAR2 and phosphorylated TF specifically on neovasculature. Overall, these observations suggest that phosphorylation of the TF cytoplasmic domain releases its negative regulatory control of angiogenesis. The role of the TF cytoplasmic domain as a negative regulator of physiological process has been further supported by the recent observation that TF expression suppresses 3?1-dependent migration on laminin 5, an effect that is reversed by PAR2-dependent phosphorylation of the TF cytoplasmic domain.76 Although these data support nonhemostatic roles for TF in angiogenesis and tumor metastasis, they do not fully explain the earlier studies that demonstrated that the TF–cytoplasmic domain contributes to cell migration,77 VEGF production,57 and tumor metastasis.54,55 The identified link between PAR2–TF and TF–integrins add yet another facet to the complex regulation of angiogenesis by TF–VIIa signaling.
Tumor Metastasis
The role of TF in metastasis is well-documented. TF expression has been correlated with malignant progression in several types of cancer.78–82 Experiments using mouse models have demonstrated that TF in tumor cells promotes hematogenous metastasis, and both the TF cytoplasmic domain and the TF–VIIa protease activity contribute to this metastasis.54–56,83 It is likely that the principle role for TF–VIIa protease activity in tumor metastasis is to generate thrombin and fibrin, which could play a direct role in tumor metastasis. Nonetheless, there is a possibility that TF–VIIa signaling may also contribute to tumor metastasis because it alters many cellular processes that are associated with tumor metastasis, such as angiogenesis. Furthermore, FVIIa induces the activation of both p42/44 MAPK and protein kinase B pathways, 2 pathways known to inhibit apoptosis.84,85 Resistance to apoptosis is a key factor in the survival of malignant cells. Defects in apoptosis or activation of anti-apoptotic pathways promote tumor growth and survival, which may be linked to tumor metastasis.86,87 Recent studies88,89 have shown that TF–VIIa activates anti-apoptotic signaling pathways in serum-deprived cells, thereby increasing their survival. Additionally, FVIIa was found to inhibit apoptosis induced by the loss of adhesion.88 Therefore, TF–VIIa-induced cell survival, in addition to other TF–VIIa-induced cellular processes, may contribute to tumor growth and metastasis. However, it should be noted that the evidence for the anti-apoptotic effect of FVIIa is limited to cells that were transfected to overexpress TF. Confirmation of the anti-apoptotic effect of FVIIa in tumor cells and/or stromal cells surrounding a tumor is essential before strong mechanistic conclusions of how TF–VIIa contributes to tumor growth and metastasis can be drawn.
Although it is logical to assume that tumor TF contributes to tumor growth and metastasis through TF–VIIa activation of PAR2 and/or PAR1, currently there is no evidence to support this. Both PAR1 and PAR2 are coexpressed in tumor cells and cells surrounding a tumor in the tumor microenvironment.90 Although many studies document the importance of thrombin activation of PAR1 in tumor cells in metastasis,91–97 little is known regarding the role of tumor cell PAR2 in metastasis, let alone the importance of TF–VIIa-induced activation of PAR2. Recently, Shi et al98 showed that both PAR1 and PAR2 are involved in tumor metastasis and PAR2 effects on tumor cell migration and metastasis are thrombin-dependent. At present, it is unclear whether PAR2 in metastasis is activated indirectly by thrombin or directly by a protease with trypsin-like activity, such as FVIIa. Studies addressing the role of PAR1 and PAR2 of host tissues in tumor metastasis show both PAR1 and PAR2 deficiency have no effect on tumor metastasis.99 In contrast, genetic deficiency in platelet production or activation protected mice against metastasis in hematogenous metastasis model system.99 These data raise a valid question whether PAR1 and PAR2 activation in endothelial or inflammatory cells contribute to tumor metastasis.
Wound Healing
The epidermis is a rich source for TF.1 Most wounds to the skin will invariably cause leakage of blood from the damaged blood vessels. This leakage allows the formation of a fibrin clot, which temporarily shields the wound and protects the denuded wound tissues, and it also releases various growth factors and cytokines from degranulating platelets that trigger the repair process immediately after injury.100,101 The blood leakage at the wound site will also allow FVIIa binding to TF on keratinocytes, raising a possibility for a role for TF–VIIa-induced cell signaling in the wound repair. Consistent with this scenario, Camerer et al28 showed that the interaction of FVIIa with TF on human keratinocytes upregulated a number of genes that are shown to be involved in the early steps of wound healing. They include transcription regulators (c-fos, EGR-1, ETR101, BTEB2, c-myc, fra-1, and tristeraproline), growth factors (amphiregulin, hbEGF, CCN2, and FGF-5), pro-inflammatory cytokines (IL-1?, IL-8, LIF, and MIP2), proteins involved in cellular reorganization/migration (RhoE, uPAR, and collagenases 1 and 3), and others (plasminogen activator inhibitor-2, cyclophilin, GADD45, Jagged 1, and prostaglandin E2 receptor). Because FVIIa binding to TF would occur immediately on wounding, TF–VIIa signaling may provide an early signal in the wound repair process. The expression pattern of TF–VIIa-induced genes suggests that TF–VIIa signaling may contribute to various steps in wound healing. TF–VIIa signaling may also play a role in wound healing indirectly through elaboration of gene products that in turn could induce cell signaling appropriate for the wound repair process. In this context, TF–VIIa-induced expression of CCN1 and CCN2 in keratinocytes and fibroblasts are noteworthy.28,39 CCN1 is inducibly expressed in granulation tissue during wound repair and activates genes that play multiple and coordinated roles in the wound healing process, including angiogenesis, inflammation, and extracellular matrix remodeling.102 Similarly, CCN2 contributes to wound healing via selective modulation of fibroblasts proliferation and changes to gene expression.103
Atherosclerosis and Smooth Muscle Cells
It is well-established that exposure of TF to circulating blood on rupture of atherosclerotic plaque plays an important role in the pathogenesis of thrombus formation at sites of plaque rupture, resulting in acute coronary events and myocardial infarction.104–107 Recent studies indicate that TF–VIIa signaling may play a role in the pathogenesis of atherosclerosis. TF–VIIa has been shown to be a strong chemotactic stimulus for smooth muscle cells,108 and overexpression of tissue factor pathway inhibitor in smooth muscle cells was shown to attenuate the TF–VIIa-induced cell migration.109 Consistent with this in vitro observation, overexpression of tissue factor pathway inhibitor was found to attenuate vascular remodeling in a murine model system.110 TF–VIIa-induced cell signaling has also been shown to promote platelet-derived growth factor BB-induced cell migration in fibroblasts.111 Additionally, TF–VIIa-induced cell signaling recently has been shown to lead to smooth muscle cell proliferation.112 Although our recent data113 using fibroblasts support this observation, the proliferative effect of TF–VIIa-induced cell signaling was very modest. Moreover, other studies failed to demonstrate the proliferative effect of TF–VIIa signaling.29,89,114 Therefore, further studies are needed to determine whether TF–VIIa-induced cell proliferation contributes to the pathogenesis of atherosclerosis. Because TF–VIIa-induced cell signaling in various cell types was shown to induce a number of gene products that are relevant to cell proliferation and migration, it is possible that TF–VIIa signaling may also produce similar effects in TF-expressing cells in the atherosclerosis plaques. It is of particular interest to note that TF–VIIa signaling upregulates the expression of CCN1 and CCN2 in fibroblasts.39 Similar to TF expression, CCN1 mRNA is undetectable in normal blood vessels but overexpressed in atherosclerotic lesions, primarily in vascular smooth muscle cells.115 In atherosclerosis, high levels of CCN2 expression is thought to be responsible for extracellular matrix accumulation and thus progression of atherosclerotic lesions.116 Similar to CCN2, CCN1 expression was shown to be upregulated in atherosclerotic lesions of apoE–/– mice117 and human atherosclerotic lesions.118 Because recent studies show extrahepatic synthesis of FVII in human atherosclerotic vessels,119 it is possible that TF–VIIa may induce the expression of CCN1 and CCN2 within the plaque, which could accelerate intimal thickening by promoting proliferation and migration of fibroblasts and smooth muscle cells. Further, removal or retraction of endothelial cells of atherosclerotic plaques would expose CCN1 and CCN2 in the underlying subendothelial matrix to which activated platelets and monocytes could adhere.117,120 This, in combination with TF–VIIa-induced fibrin clot, could lead to an acute arterial occlusion on rupture of the atherosclerotic plaque. Paradoxically, increased expression of CCN2 could also play a beneficial role. For example, increased expression of CCN2 along the fibrous cap may reduce the risk of plaque rupture by stabilizing the fibrous cap with extracellular matrix.
Summary and Future Directions
Recent studies suggest that TF plays a nonhemostatic role in many biological processes, and at least some of these effects are mediated via TF–VIIa-induced cell signaling. It is now established that TF–VIIa induces cell signaling via activation of PAR2 and that the ternary complex of TF–VIIa–Xa is a more efficient signaling mediator than the binary TF–VIIa complex, particularly at low concentrations of FVIIa. However, it is currently unclear whether the ternary and the binary complexes activate the same signaling pathways or if each has its own specificities. Because TF–VIIa complex primarily activates PAR2 while TF–VIIa–Xa activates both PAR1 and PAR2, it is expected that there would be some distinction between the signaling mediated by these complexes. Identification of such differences would further reinforce the importance of TF–VIIa signaling. In addition to PARs, other cell surface components, such as integrins, proteoglycans, and growth factor receptors, may also be involved in transmitting or regulating TF–VIIa signaling. Analysis of other potential cell surface receptors that may mediate TF–VIIa signaling is an important area requiring further study. The ability of FVIIa to induce cell signaling is dependent not only on the availability of PARs and TF but also on their spatial proximity. A spatial disconnection between TF and PAR2 may explain why FVIIa fails to induce signaling in some cells, even if they express both TF and PAR2. Currently, there is no information on how the spatial organization of participating signaling components regulates TF–VIIa signaling. This is also another important area in need of further investigation. Additionally, analysis of if and how phosphorylation of the TF cytoplasmic domain regulates TF–VIIa protease-induced signaling also requires future attention. Until now, the TF–VIIa-induced cell signaling studies are primarily limited to in vitro cell model systems and there is no convincing evidence that TF–VIIa signaling actually plays a role in vivo. Thus, the main challenge for future investigators is to establish the importance of TF–VIIa-induced cell signaling in pathophysiological processes in which multiple protease-induced signaling pathways operate simultaneously and redundantly in PAR-mediated signaling. The development of specific inhibitors that suppress TF–VIIa signaling but not TF–VIIa coagulant function or vice versa will provide unique opportunities to design specific drugs that may have therapeutic value in the treatment of diseases that are often associated with aberrant expression of TF.
Acknowledgments
The authors are thankful to Drs Lars Petersen, Brit Sorensen, and Mirella Ezban at Novo-Nordisk, Denmark, for their collaboration on the work related to TF–VIIa signaling. The authors’ work related to this article was supported by National Institutes of Health grants HL65550 and HL58869, and grants from American Heart Association (National and Texas affiliate).
Received September 28, 2004; accepted November 12, 2004.
References
Fleck RA, Rao LVM, Rapaport SI, Varki N. Localization of human tissue factor antigen by immunostaining with monospecific, polyclonal anti-human tissue factor antibody. Thromb Res. 1990; 59: 421–437.
Drake TA, Morrissey JH, Edgington TS. Selective cellular expression of tissue factor in human tissues: Implications for disorders of hemostasis and thrombosis. Am J Pathol. 1989; 134: 1087–1097.
Osterud B. Tissue factor expression by monocytes: regulation and pathophysiological role. Blood Coag Fibrinol. 1998; 9: S9–S14.
Contrino J, Hair G, Kreutzer DL, Rickles FR. In situ detection of tissue factor in vascular endothelial cells: Correlation with the malignant phenotype of human breast disease. Nat Med. 1996; 2: 209–215.
Luther T, Flossel C, Albrecht S, Kotzsch M, Muller M. Tissue factor expression in normal and abnormal mammary gland. Nat Med. 1996; 2: 491–492.
McNamara CA, Sarenbock IJ, Gimple LW, Fenton II JW, Coughlin SR, Owens GK. Thrombin stimulates proliferation of cultured rat aortic smooth muscle cells by a proteolytically activated receptor. J Clin Invest. 1993; 91: 94–98.
Gasic GP, Arenas CP, Gasic TB, Gasic GJ. Coagulation factors X, Xa, and protein S as potent mitogens of cultured aortic smooth muscle cells. Proc Natl Acad Sci U S A. 1992; 89: 2317–2320.
Coughlin SR. Protease-activated receptors in vascular biology. Thromb Haemost. 2001; 86: 298–307.
Pendurthi UR, Rao LVM. Factor VIIa/tissue factor-induced signaling: A link between clotting and disease. In: Litwick G, ed. Vitamins and Hormones. San Diego: Academic Press; 2002: 323–355.
Ruf W, Mueller BM. Tissue factor in cancer angiogenesis and metastasis. Curr Opin Hematol. 1996; 3: 379–384.
Mackman N. Role of tissue factor in hemostasis, thrombosis, and vascular development. Arterioscler Thromb Vasc Biol. 2004; 24: 1015–1022.
Riewald M, Kravchenko VV, Petrovan R, O’brien PJ, Brass LF, Ulevitch RJ, Ruf W. Gene induction by coagulation factor Xa is mediated by activation of PAR-1. Blood. 2001; 97: 3109–3116.
Riewald M, Petrovan RJ, Donner A, Ruf W. Activated protein C signals through the thrombin receptor PAR1 in endothelial cells. J Endotoxin Res. 2003; 9: 317–321.
Pendurthi UR, Ngyuen M, Andrade-Gordon P, Petersen LC, Rao LVM. Plasmin induces Cyr61 gene expression in fibroblasts via protease activated receptor-1 and p44/42 mitogen-activated protein kinase-dependent signaling pathway. Arterioscler Thromb Vasc Biol. 2002; 22: 1421–1426.
Camerer E, Huang W, Coughlin SR. Tissue factor- and factor X-dependent activation of protease-activated receptor-2 by factor VIIa. Proc Natl Acad Sci U S A. 2000; 97: 5255–5260.
Bono F, Schaeffer P, Herault J-P, Michaux C, Nestor A-L, Guillemot J-C, Herbert JM. Factor Xa activates endothelial cells by a receptor cascade between EPR-1 and PAR-2. Arterioscler Thromb Vasc Biol. 2000; 20: e107–e112.
Coughlin SR. Thrombin signalling and protease-activated receptors. Nature. 2000; 407: 258–264.
Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, Plevin R. Proteinase-activated receptors. Pharmacol Rev. 2001; 53: 245–282.
O’brien PJ, Molino M, Kahn M, Brass LF. Protease activated receptors: theme and variations. Oncogene. 2001; 20: 1570–1581.
Bazan JF. Structural design and molecular evolution of a cytokine receptor super family. Proc Natl Acad Sci U S A. 1990; 87: 6934–6938.
Rottingen JA, Enden T, Camerer E, Iversen JG, Prydz H. Binding of human factor VIIa to tissue factor induces cytosolic Ca2+ signals in J82 cells, transfected COS-1 cells, Madin-Darby canine kidney cells and in human endothelial cells induced to synthesize tissue factor. J Biol Chem. 1995; 270: 4650–4660.
Camerer E, Rottingen JA, Iversen JG, Prydz H. Coagulation factors VII and X induce Ca2+ oscillations in Madin-Darby canine kidney cells. J Biol Chem. 1996; 271: 29034–29042.
Prydz HS, Camerer E, Rottingen J-A, Wiiger MT, Gjernes E. Cellular consequences of the initiation of blood coagulation. Thromb Haemost. 1999; 82: 183–192.
Camerer E, Rottingen J-A, Gjernes E, Larsen K, Skartlien AH, Iversen J-G, Prydz H. Coagulation factors VIIa and Xa induce cell signaling leading to up-regulation of the egr-1 gene. J Biol Chem. 1999; 274: 32225–32233.
Poulsen LK, Jacobsen N, Sorensen BB, Bergenhem NCH, Kelly JD, Foster DC, Thastrup O, Ezban M, Petersen LC. Signal transduction via the mitogen-activated protein kinase pathway induced by binding of coagulation factor VIIa to tissue factor. J Biol Chem. 1998; 273: 6228–6232.
Sorensen BB, Freskgard P-O, Nielsen LS, Rao LVM, Ezban M, Petersen LC. Factor VIIa-induced p44/42 mitogen-activated kinase activation requires the proteolytic activity of factor VIIa and is independent of the tissue factor cytoplasmic domain. J Biol Chem. 1999; 274: 21349–21354.
Pendurthi U, Sorensen BB, Petersen LC, Ezban M, Hagel G, Thastrup O, Rao LVM. Factor VIIa induced signaling in fibroblasts. Blood. 2000; 96: 635a.
Camerer E, Gjernes E, Wiiger M, Pringle S, Prydz H. Binding of factor VIIa to tissue factor on keratinocytes induces gene expression. J Biol Chem. 2000; 275: 6580–6585.
Versteeg HH, Hoedemaeker I, Diks SH, Stam JC, Spaargaren M, Bergen en Henegouwen PMP, Deventer SJH, Peppelenbosch MP. Factor VIIa/Tissue factor-induced signaling via activation of Src-like Kinase, Phosphatidylinositol 3-Kinase, and Rac. J Biol Chem. 2000; 275: 28750–28756.
Versteeg HH, Bresser HL, Spek CA, Richel DJ, Deventer SJH. Regulation of the p21 RAS-MAP kinase pathway by factor VIIa. J Thromb Haemost. 2003; 1: 1012–1018.
Wiiger MT, Prydz H. The epidermal growth factor receptor (EGFR) and proline rich tyrosine kinase 2 (PYK2) are involved in tissue factor dependent factor VIIa signalling in HaCaT cells. Thromb Haemost. 2004; 92: 13–22.
Lev S, Moreno H, Martinez R, Canoll P, Peles E, Musacchio JM, Plowman GD, Rudy B, Schlessinger J. Protein tyrosine kinase PYK2 involved in Ca(2+)-induced regulation of ion channel and MAP kinase functions. Nature. 1995; 376: 737–745.
Versteeg HH, Spek CA, Slofstra SH, Diks SH, Richel DJ, Peppelenbosch MP. FVIIa: TF induces cell survival via G12/G13-dependent Jak/STAT activation and BclXL production. Circ Res. 2004; 94: 1032–1040.
Versteeg HH, Sorensen BB, Slofstra S, Van den Brande JHM, Stam JC, van Bergen en Henegouwen PMP, Richel DJ, Petersen LC, Peppelenbosch MP. VIIa/tissue factor interaction results in a tissue factor cytoplasmic domain-independent activation of protein synthesis, p70, and p90 S6 kinase phosphorylation. J Biol Chem. 2002; 277: 27065–27072.
Ollivier V, Bentolila S, Chabbat J, Hakim J, de Prost D. Tissue factor-dependent vascular endothelial growth factor production by human fibroblasts in response to activated factor VII. Blood. 1998; 91: 2698–2703.
Taniguchi T, Kakkar AK, Tuddenham EGD, Williamson RCN, Lemoine NRL. Enhanced expression of urokinase receptor induced through the tissue factor-factor VIIa pathway in human pancreatic cancer. Cancer Res. 1998; 58: 4461–4467.
Wang X, Gjernes E, Prydz H. Factor VIIa induces tissue factor-dependent up-regulation of interleukin-8 in a human keratinocyte line. J Biol Chem. 2002; 277: 23620–23626.
Hjortoe GM, Petersen LC, Albrektsen T, Sorensen BB, Norby PL, Mandal SK, Pendurthi UR, Rao LV. Tissue factor-factor VIIa-specific up-regulation of IL-8 expression in MDA-MB-231 cells is mediated by PAR-2 and results in increased cell migration. Blood. 2004; 103: 3029–3037.
Pendurthi UR, Allen KE, Ezban M, Rao LVM. Factor VIIa and thrombin induce the expression of Cyr61 and connective tissue growth factor, extracellular matrix signaling proteins that could act as possible downstream mediators in factor VII. tissue factor-induced signal transduction. J Biol Chem. 2000; 275: 14632–14641.
Lau LF, Lam S. The CCN family of angiogenic regulators: The integrin connection. Exp Cell Res. 1999; 248: 44–57.
Petersen LC, Thastrup O, Hagel G, Sorensen BB, Freskgard P-O, Rao LVM, Ezban M. Exclusion of known protease activated receptors in factor VIIa-induced signal transduction. Thromb Haemost. 2000; 83: 571–576.
Riewald M, Ruf W. Mechanistic coupling of protease signaling and initiation of coagulation by tissue factor. Proc Natl Acad Sci U S A. 2001; 98: 7742–7747.
Marutsuka K, Hatakeyama K, Sato Y, Yamashita A, Sumiyoshi A, Asada Y. Protease-activated receptor -2 (PAR2) mediates vascular smooth muscle cell migration induced by tissue factor/factor VIIa complex. Thromb Res. 2002; 107: 271–276.
Ishii K, Hein L, Kobilka B, Coughlin SR. Kinetics of thrombin receptor cleavage on intact cells. Relation to signaling. J Biol Chem. 1993; 268: 9780–9785.
Morrissey JH, Macik BG, Neuenschwander PF, Comp PC. Quantitation of activated factor VII levels in plasma using a tissue factor mutant selectively deficient in promoting factor VII activation. Blood. 1993; 81: 734–744.
Wildgoose P, Nemerson Y, Hansen LL, Nielsen FE, Glazer S, Hedner U. Measurement of basal levels of factor VIIa in hemophilia A and B patients. Blood. 1992; 80: 25–28.
Neuenschwander PF, Fiore MM, Morrissey JH. Factor VII autoactivation proceeds via interaction of distinct protease-cofactor and zymogen-cofactor complexes. J Biol Chem. 1993; 268: 21489–21492.
Yamamoto M, Nakagaki T, Kisiel W. Tissue factor dependent autoactivation of human blood coagulation factor VII. J Biol Chem. 1992; 267: 19089–19094.
Rao LVM, Williams T, Rapaport SI. Studies of the activation of factor VII bound to tissue factor. Blood. 1996; 87: 3738–3748.
Ruf W, Dorfleutner A, Riewald M. Specificity of coagulation factor signaling. J Thromb Haemost. 2003; 1: 1495–1503.
Zioncheck TF, Roy S, Vehar GA. The cytoplasmic domain of tissue factor is phosphorylated by a protein kinase C-dependent mechanism. J Biol Chem. 1992; 267: 3561–3564.
Mody RS, Carson SD. Tissue factor cytoplasmic domain peptide is multiply phosphorylated in vitro. Biochem. 1997; 36: 7869–7875.
Dorfleutner A, Ruf W. Regulation of tissue factor cytoplasmic domain phosphorylation by palmitoylation. Blood. 2003; 102: 3998–4005.
Bromberg ME, Konigsberg WH, Madison JF, Pawashe A, Garen A. Tissue factor promotes melanoma metastasis by a pathway independent of blood coagulation. Proc Natl Acad Sci U S A. 1995; 92: 8205–8209.
Mueller BM, Ruf W. Requirement for binding of catalytically active factor VIIa in tissue factor-dependent experimental metastasis. J Clin Invest. 1998; 101: 1372–1378.
Bromberg ME, Sundaram R, Homer RJ, Garen A, Konigsberg WH. Role of tissue factor in metastasis: Functions of the cytoplasmic and extracellular domains of the molecule. Thromb Haemost. 1999; 82: 88–92.
Abe K, Shoji M, Chen J, Bierhaus A, Danave I, Micko C, Casper K, Dillehay DL, Nawroth P, Rickles FR. Regulation of vascular endothelial growth factor production and angiogenesis by the cytoplasmic tail of tissue factor. Proc Natl Acad Sci U S A. 1999; 96: 8663–8668.
Bromberg ME, Garen A, McNiff J, Konigsberg WH. Overexpression of tissue factor does not promote metastasis by upregulation of VEGF or angiogenesis in melanoma cells. Blood. 1997; 90 (suppl): 293a.
Ahamed J, Ruf W. Protease-activated receptor 2-dependent phosphorylation of the tissue factor cytoplasmic domain. J Biol Chem. 2004; 279: 23038–23044.
Belting M, Dorrell MI, Sandgren S, Aguilar E, Ahamed J, Dorfleutner A, Carmeliet P, Mueller BM, Friedlander M, Ruf W. Regulation of angiogenesis by tissue factor cytoplasmic domain signaling. Nat Med. 2004; 10: 502–509.
Taylor FB, Chang A, Ruf W, Morrissey JH, Hinshaw LB, Catlett R, Blick K, Edgington TS. Lethal E. coli septic shock is prevented by blocking tissue factor with monoclonal antibody. Circ Shock. 1991; 33: 127–134.
Creasey AA, Chang ACK, Feigen L, Wun TC, Taylor FB, Hinshaw LB. Tissue factor pathway inhibitor reduces mortality from E. coli septic shock. J Clin Invest. 1993; 91: 2850–2860.
Taylor FB, Chang ACK, Peer G, Li A, Ezban M, Hedner U. Active site inhibited factor VIIa (DEGR VIIa) attenuates the coagulant and interleukin-6 and -8 but not tumor necrosis factor responses of the baboon to LD 100 E. coli. Blood. 1998; 91: 1609–1615.
Taylor FB, Chang ACK, Peer GT, Mather T, Blick K, Catlett R, Lockhart M, Esmon CT. DEGR-factor Xa blocks disseminated intravascular coagulation initiated by E. Coli without preventing shock or organ damage. Blood. 1991; 78: 364–368.
Randolph MM, White GL, Kosanke SD, Bild G, Carr C, Galluppi G, Hinshaw LB, Taylor FB, Jr. Attenuation of tissue thrombosis and hemorrhage by ala-TFPI does not account for its protection against E. coli–a comparative study of treated and untreated non-surviving baboons challenged with LD100 E. coli. Thromb Haemost. 1998; 79: 1048–1053.
Miller DL, Welty-wolf KE, Carraway MS, Ezban M, Ghio A, Suliman H, Piantadosi CA. Extrinsic coagulation cascade blockade attenuates lung injury and proinflammatory cytokine release after intratracheal lipopolysaccharide. Am J Respir Cell Mol Biol. 2002; 26: 650–658.
Randolph GJ, Luther T, Albrecht S, Magdolen V, Muller WA. Role of tissue factor in adhesion of mononuclear phagocytes to and trafficking through endothelium in vitro. Blood. 1998; 92: 4167–4177.
Cunningham MA, Romas P, Hutchinson P, Holdsworth SR, Tipping PG. Tissue factor and factor VIIa receptor/ligand interactions induce proinflammatory effects in macrophages. Blood. 1999; 94: 3413–3420.
Pawlinski R, Pedersen B, Schabbauer G, Tencati M, Holscher T, Boisvert W, Andrade-Gordon P, Frank RD, Mackman N. Role of tissue factor and protease-activated receptors in a mouse model of endotoxemia. Blood. 2004; 103: 1342–1347.
Sharma L, Melis E, Hickey MJ, Clyne CD, Erlich J, Khachigian LM, Davenport P, Morand E, Carmeliet P, Tipping PG. The cytoplasmic domain of tissue factor contributes to leukocyte recruitment and death in endotoxemia. Am J Pathol. 2004; 165: 331–340.
Browder T, Folkman J, Pirie-Shepherd S. The hemostatic system as a regulator of angiogenesis. J Biol Chem. 2000; 275: 1521–1524.
Zhang Y, Deng Y, Luther T, Muller M, Ziegler R, Waldherr R, Stern DM, Nawroth P. Tissue factor controls the balance of angiogenic and antiangiogenic properties of tumor cells in mice. J Clin Invest. 1994; 94: 1320–1327.
Li A, Dubey S, Varney ML, Dave BJ, Singh RK. IL-8 directly enhanced endothelial cell survival. proliferation, and matrix metalloproteinases production and regulated angiogenesis. J Immunol. 2003; 170: 3369–3376.
Kireeva ML, Lam S, Lau LF. Adhesion of human umbilical vein endothelial cells to the immediate-early gene product cyr61 is mediated through integrin v?3. J Biol Chem. 1998; 273: 3090–3096.
Planque N, Perbal B. A structural approach to the role of CCN (CYR61/CTGF/NOV) proteins in tumourigenesis. Cancer Cell Int. 2003; 3: 15.
Dorfleutner A, Hintermann E, Tarui T, Takada Y, Ruf W. Crosstalk of Integrin {alpha}3{beta}1 and Tissue Factor in Cell Migration. Mol Biol Cell. 2004; 15: 4416–4425.
Ott I, Fischer EG, Miyagi Y, Mueller BM, Ruf W. A role for tissue factor in cell adhesion and migration mediated by interaction with actin-binding protein 280. J Cell Biol. 1998; 140: 1241–1253.
Rao LVM. Tissue factor as a tumor procoagulant. Cancer Metastasis Rev. 1992; 11: 249–266.
Hamada K, Kuratsu J, Saitoh Y, Nishi T, Ushio Y. Expression of tissue factor expression correlates with grade of malignancy in human glioma. Cancer. 1996; 77: 1877–1883.
Kakkar AK, Lemoine NR, Scully MF, Tebbutt S, Williamson RC. Tissue factor expression correlates with histological grade in human pancreatic cancer. Br J Surg. 1995; 82: 1101–1104.
Shigemori C, Wada H, Matsumoto K, Shiku H, Nakamura S, Suzuki H. Tissue factor expression and metastatic potential of colorectal cancer. Thromb Haemost. 1998; 80: 894–898.
Seto S, Onodera H, Kaido T, Yoshikawa A, Ishigami S, Arii S, Imamura M. Tissue factor expression in human colorectal carcinoma: correlation with hepatic metastasis and impact on prognosis. Cancer. 2000; 88: 295–301.
Mueller BM, Reisfeld RA, Edgington TS, Ruf W. Expression of tissue factor by melanoma cells promotes efficient hematogenous metastasis. Proc Natl Acad Sci U S A. 1992; 89: 11832–11836.
Dimmeler S, Zeiher AM. Endothelial cell apoptosis in angiogenesis and vessel regression. Circ Res. 2000; 87: 434–439.
Bergmann A, Agapite J, McCall K, Steller H. The Drosophila gene hid is a direct molecular target of Ras-dependent survival signaling. Cell. 1998; 95: 331–341.
Yu J, Zhang L. Apoptosis in human cancer cells. Curr Opin Oncol. 2004; 16: 19–24.
Debatin KM. Apoptosis pathways in cancer and cancer therapy. Cancer Immunol Immunother. 2004; 53: 153–159.
Versteeg HH, Spek CA, Richel DJ, Peppelenbosch MP. Coagulation factors VIIa and Xa inhibit apoptosis and anoikis. Oncog. 2004; 23: 410–417.
Sorensen BB, Rao LVM, Tornehave D, Gammeltoft S, Petersen LC. Antiapoptotic effect of coagulation factor VIIa. Blood. 2003; 102: 1708–1715.
D’Andrea MR, Derian CK, Santulli RJ, Andrade-Gordon P. Differential expression of protease-activated receptors-1 and -2 in stromal fibroblasts of normal, benign, and malignant human tissues. Am J Pathol. 2001; 158: 2031–2041.
Even-ram SC, Uziely B, Cohen P, Grisaru-granovsky S, Reich R, Bar-Shavit R, Giocondi M. Thrombin receptor overexpression in malignant and physiological invasion processes. Nat Med. 1998; 4: 909–914.
Fischer EG, Ruf W, Mueller BM. Tissue factor-initiated thrombin generation activates the signalling thrombin receptor on malignant melanoma cells. Cancer Res. 1995; 55: 1629–1632.
Tellez C, Bar-Eli M. Role and regulation of the thrombin receptor (PAR-1) in human melanoma. Oncogene. 2003; 22: 3130–3137.
Nierodzik ML, Chen K, Takeshita K, Li JJ, Huang YQ, Feng XS, D’Andrea MR, Andrade-Gordon P, Karpatkin S. Protease-activated receptor 1 (PAR-1) is required and rate-limiting for thrombin-enhanced experimental pulmonary metastasis. Blood. 1998; 92: 3694–3700.
Bromberg ME, Bailly MA, Konigsberg WH. Role of protease-activated receptor 1 in tumor metastasis promoted by tissue factor. Thromb Haemost. 2001; 86: 1210–1214.
Even-ram SC, Maoz M, Pokroy E, Reich R, Katz B-Z, Gutwein P, Altevogt P, Bar-Shavit R. Tumor cell invasion is promoted by activation of protease activated receptor-1 in cooperation with the v?5 integrin. J Biol Chem. 2001; 276: 10952–10956.
Darmoul D, Gratio V, Devaud H, Lehy T, Laburthe M. Aberrant expression and activation of the thrombin receptor protease-activated receptor-1 induces cell proliferation and motility in human colon cancer cells. Am J Pathol. 2003; 162: 1503–1513.
Shi X, Gangadharan B, Brass LF, Ruf W, Mueller BM. Protease-activated receptors (PAR1 and PAR2) contribute to tumor cell motility and metastasis. Mol Cancer Res. 2004; 2: 395–402.
Camerer E, Qazi AA, Duong DN, Cornelissen I, Advincula R, Coughlin SR. Platelets, protease-activated receptors, and fibrinogen in hematogenous metastasis. Blood. 2004; 104: 397–401.
Werner S, Grose R. Regulation of wound healing by growth factors and cytokines. Physiol Rev. 2003; 83: 835–870.
Martin P. Wound healing–aiming for perfect skin regeneration. Science. 1997; 276: 75–81.
Chen C-C, Mo F-E, Lau LF. The angiogenic factor Cyr61 activates a genetic program for wound healing in human skin fibroblasts. J Biol Chem. 2001; 276: 47329–47334.
Wang JF, Olson ME, Ball DK, Brigstock DR, Hart DA. Recombinant connective tissue growth factor modulates porcine skin fibroblast gene expression. Wound Repair Regen. 2003; 11: 220–229.
Ardissino D, Merlini PA, Arlens R, Coppola R, Bramucci E, Lucreziotti S, Repetto A, Fetiveau R, Mannucci PM. Tissue factor in human coronary atherosclerotic plaques. Clinica Chimica Acta. 2000; 291: 235–240.
Ardissino D, Merlini PA, Bauer KA, Bramucci E, Ferrario M, Coppola R, Fetiveau R, Lucreziotti S, Rosenberg RD, Mannucci PM. Thrombogenic potential of human coronary atherosclerotic plaques. Blood. 2001; 98: 2726–2729.
Marmur JD, Thiruvikraman SV, Fyfe BS, Guha A, Sharma SK, Ambrose JA, Fallon JT, Nemerson Y, Taubman MB. Identification of active tissue factor in human coronary atheroma. Circulation. 1996; 94: 1226–1232.
Taubman MB, Fallon JT, Schecter AD, Giesen P, Mendlowitz M, Fyfe BS, Marmur JD, Nemerson Y. Tissue factor in the pathogenesis of atherosclerosis. Thromb Haemost. 1997; 78: 200–204.
Sato Y, Asada Y, Marutsuka K, Hatakeyama K. Tissue factor induces migration of cultured aortic smooth muscle cells. Thromb Haemost. 1996; 75: 389–392.
Sato Y, Kataoka H, Asada Y, Marutsuka K, Kamikubo YI, Koono M, Sumiyoshi A. Overexprssion of tissue factor pathway inhibitor in aortic smooth muscle cells inhibits cell migration induced by tissue factor/factor VIIa complex. Thrombosis Res. 1999; 94: 401–406.
Singh R, Pan S, Mueske CS, Witt T, Kleppe LS, Peterson TE, Slobodova A, Chang J-Y, Caplice NM, Simari RD. Role for tissue factor pathway in murine model of vascular remodeling. Circ Res. 2001; 89: 71–76.
Siegbahn A, Johnell M, Rorsman C, Ezban M, Heldin C-H, Ronnstrand L. Binding of factor VIIa to tissue factor on human fibroblasts leads to activation of phospholipase C and enhanced PDGF-BB-stimulated chemotaxis. Blood. 2000; 96: 3452–3458.
Cirillo P, Cali G, Golino P, Calabro P, Forte L, De Rosa S, Pacileo M, Ragni M, Scopacasa F, Nitsch L, Chiariello M. Tissue factor binding of activated factor VII triggers smooth muscle cell proliferation via extracellular signal-regulated kinase activation. Circulation. 2004; 109: 2911–2916.
Mandal SK, Rao LVM, Tran TT, Pendurthi UR. A novel mechanism of plasmin-induced mitogenesis in fibroblasts. J Thromb Haemost 2004. In press.
Herbert JM, de Prost D, Ollivier V, Melis E, Carmeliet P. Tissue factor is not involved in the mitogenic activity of factor VIIa. Biochem Biophys Res Commun. 2001; 281: 1074–1077.
Oemar BS, Werner A, Garnier JM, Do DD, Godoy N, Nauck M, Marz W, Rupp J, Pech M, Luscher TF. Human connective tissue growth factor is expressed in advanced atherosclerotic lesions. Circulation. 1997; 95: 831–839.
Oemar BS, Luscher TF. Connective tissue growth factor: Friend or foe? Arterioscler Thromb Vasc Biol. 1997; 17: 1483–1489.
Schober JM, Chen N, Grzeszkiewicz TM, Jovanovic I, Emeson EE, Ugarova TP, Ye RD, Lau LF, Lam SC. Identification of integrin alpha(M)beta(2) as an adhesion receptor on peripheral blood monocytes for Cyr61 (CCN1) and connective tissue growth factor (CCN2): immediate-early gene products expressed in atherosclerotic lesions. Blood. 2002; 99: 4457–4465.
Hilfiker A, Hilfiker-Kleiner D, Fuchs M, Kaminski K, Lichtenberg A, Rothkotter HJ, Schieffer B, Drexler H. Expression of CYR61, an angiogenic immediate early gene, in arteriosclerosis and its regulation by angiotensin II. Circulation. 2002; 106: 254–260.
Wilcox JN, Noguchi S, Casanova J. Extrahepatic synthesis of factor VII in human atherosclerotic vessels. Arterioscler Thromb Vasc Biol. 2003; 23: 136–141.
Jedsadayanmate A, Chen C-C, Kireeva ML, Lau LF. Activation-dependent adhesion of human platelets to Cyr61 and Fisp12/mouse connective tissue growth factor is mediated through integrin aIIb?3. J Biol Chem. 1999; 274: 24321–24327.(L. Vijaya Mohan Rao; Usha)