Tissue factor (TF), the cellular receptor for factor VIIa (FVIIa), besides initiating blood coagulation, is believed to play an important role in tissue repair, inflammation, angiogenesis, and tumor metastasis. Like TF, the chemokine interleukin-8 (IL-8) is shown to play a critical role in these processes. To elucidate the potential mechanisms by which TF contributes to tumor invasion and metastasis, we IntroductionCells that express tissue factor (TF) are usually not exposed to the blood. However, in normal response to vessel injury, TF exposure is an initial event of a strictly regulated process resulting in fibrin deposition, inflammation, angiogenesis, and tissue repair. Carcinomas exploit a normal physiologic response in a way that allows tumor growth and dissemination. It has long been presumed that tumors may take advantage of the hemostatic system. A relationship between increased clotting and malignancy was recognized more than a century ago. 1 Numerous clinical observations suggest that the hemostatic system is frequently activated in cancer patients. 2-5 Many tumor types have been shown to express TF. 6,7 Further, the level of TF expression in various tumor types has been shown to correlate with their metastatic potential. [8][9][10] Studies carried out with mouse tumor metastasis models establish that TF plays a critical role in tumor metastasis. 11,12 TF is the cellular receptor for coagulation factor VIIa (FVIIa). TF-induced metastasis requires participation of the cytoplasmic tail of TF and assembly of an active TF-FVIIa complex, 13,14 indicating a dual function for TF in tumor metastasis. The TF cytoplasmic domain, through its specific interaction with ABP-280, has been shown to support cell adhesion and migration. 15 At present it is unclear how TF on tumor cells contributes to tumor metastasis and whether the TF-FVIIa complex plays a direct role or whether its sole requirement is for the downstream generation of active coagulation factors, particularly thrombin, which have been implicated in tumor metastasis. [16][17][18] Recent studies show that proteolytic hydrolysis mediated by the TF-FVIIa complex induces cell signaling through G-proteincoupled receptors in a number of cell types (for reviews, see Prydz et al, 19 Pendurthi and Rao, 20 Ruf et al 21 ). TF-FVIIa-induced signaling in various cell types was shown to alter the expression of specific genes that encode transcription factors, growth factors, and proteins related to cellular reorganization. [22][23][24][25][26][27] These studies suggest that TF-FVIIa-induced signaling may play a role in growthpromoting settings, such as wound healing and cancer. However, it has yet to be shown how TF-FVIIa-induced regulation of gene expression actually affects cell phenotype or pathophysiologic processes. Moreover, a considerable overlap in signaling induced by TF-FVIIa and various other proteases, especially a highmagnitude response generated by thrombin, raises a valid question about the potential significance of TF-FVIIa-induced signaling in path...

AbstractHemophilia is treated by IV replacement therapy with Factor VIII (FVIII) or Factor IX (FIX), either on demand to resolve bleeding, or as prophylaxis. Improved treatment may be provided by drugs designed for subcutaneous and less frequent administration with a reduced risk of inhibitor formation. Tissue factor pathway inhibitor (TFPI) down-regulates the initiation of coagulation by inhibition of Factor VIIa (FVIIa)/tissue factor/Factor Xa (FVIIa/TF/FXa). Blockage of TFPI inhibition may facilitate thrombin generation in a hemophilic setting. A high-affinity (KD = 25pM) mAb, mAb 2021, against TFPI was investigated. Binding of mAb 2021 to TFPI effectively prevented inhibition of FVIIa/TF/FXa and improved clot formation in hemophilia blood and plasma. The binding epitope on the Kunitz-type protease inhibitor domain 2 of TFPI was mapped by crystallography, and showed an extensive overlap with the FXa contact region highlighting a structural basis for its mechanism of action. In a rabbit hemophilia model, an intravenous or subcutaneous dose significantly reduced cuticle bleeding. mAb 2021 showed an effect comparable with that of rFVIIa. Cuticle bleeding in the model was reduced for at least 7 days by a single intravenous dose of mAb 2021. This study suggests that neutralization of TFPI by mAb 2021 may constitute a novel treatment option in hemophilia.

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The putative role of tissue factor (TF) as a receptor involved in signal transduction is indicated by its sequence homology to cytokine receptors (Bazan, J. F. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6934-6938). Signal transduction induced by binding of FVIIa to cells expressing TF was studied with baby hamster kidney (BHK) cells stably transfected with TF and with a reporter gene construct encoding a luciferase gene under transcriptional control of tandem cassettes of signal transducer and activator of transcription (STAT) elements and one serum response element (SRE). FVIIa induced a significant luciferase response in cells expressing TF, BHK(+TF), but not in cells without TF. The BHK(+TF) cells responded to the addition of FVIIa in a dose-dependent manner, whereas no response was observed with active site-inhibited FVIIa, which also worked as an antagonist to FVIIa-induced signaling. Activation of the p44/42 MAPK pathway upon binding of FVIIa to TF was demonstrated by suppression of signaling with the specific kinase inhibitor PD98059 and demonstration of a transient p44/42 MAPK phosphorylation. No stimulation of p44/42 MAPK phosphorylation was observed with catalytically inactive FVIIa derivatives suggesting that the catalytic activity of FVIIa was obligatory for activation of the MAPK pathway. Signal transduction caused by a putative generation of FXa activity was excluded by experiments showing that FVIIa/TF-induced signaling was not quenched by tick anticoagulant protein, just as addition of FXa could not induce phosphorylation of p44/42 MAPK in BHK(+TF) cells. These results suggest a specific mechanism by which binding of FVIIa to cell surface TF independent of coagulation can modulate cellular functions and possibly play a role in angiogenesis and tumor metastasis as indicated by several recent observations.

Current management of hemophilia B entails multiple weekly infusions of factor IX (FIX) to prevent bleeding episodes. In an attempt to make a longer acting recombinant FIX (rFIX), we have explored a new releasable protraction concept using the native N-glycans in the activation peptide as sites for attachment of polyethylene glycol (PEG). Release of the activation peptide by physiologic activators converted glycoPEGylated rFIX (N9-GP) to native rFIXa and proceeded with normal kinetics for FXIa, while the K m for activation by FVIIa-tissue factor (TF) was increased by 2-fold. Consistent with minimal perturbation of rFIX by the attached PEG, N9-GP retained 73%-100% specific activity in plasma and whole-blood-based assays and showed efficacy comparable with rFIX in stopping acute bleeds in hemophilia B mice. In animal models N9-GP exhibited up to 2-fold increased in vivo recovery and a markedly prolonged half-life in mini-pig (76 hours) and hemo- IntroductionFactor IX (FIX) is a vitamin K-dependent glycoprotein and an essential protease of the hemostatic system. The domain organization of FIX is shared with factors VII, X, and protein C and comprises an N-terminal domain rich in ␥-carboxyglutamic acid (Gla), 2 epidermal growth factor-like repeats and a C-terminal trypsin-like protease domain. 1 Together they form a 55-kDa single-chain protease precursor circulating in plasma at a concentration of approximately 90nM (5 g/mL), defined as 1 IU/mL. FIX is converted to the 2-chain activated form by the tissue factor (TF)-factor VIIa (FVIIa) complex or factor XIa (FXIa). Activation occurs by limited proteolysis at Arg145 and Arg180 in the protease domain and liberates a 35-amino acid activation peptide that carries the only 2 N-linked glycans in the protein. 2,3 Subsequent assembly of FIXa with the cofactor VIIIa on the activated platelet surface greatly enhances the proteolytic activity of FIXa toward its substrate factor X (FX) and is essential for propagation of the coagulation response. 4 The importance of this activity is reflected by the occurrence of the bleeding disorder hemophilia B (HB) in individuals carrying mutations in the FIX gene. The prevalence of HB is approximately 1 in 25 000 males, and it has been estimated that approximately 84 000 people are affected worldwide. 5 The mainstay in HB treatment is substitution therapy by infusion of plasma-derived or recombinant FIX (rFIX). The therapeutic goal is to prevent bleeding episodes and to provide safe and efficacious treatment of bleedings when they occur. Because of the relatively short half-life of FIX (18-24 hours [6][7][8] ), the recommended prophylaxis regimen consists of 2 to 3 weekly infusions of 40-100 IU/kg 9 FIX to maintain trough levels above 1% and thus shifting patients from a severe to a milder phenotype. When adhered to, prophylaxis in patients without severe joint disorder is efficacious with a frequency of only 0-2 breakthrough bleeds per year in the majority of patients. 8,10 However, the need for multiple weekly infusions present challen...

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IntroductionLocal and systemic coagulation activation is a hallmark of advanced malignancies. 1,2 Cancer cell-expressed tissue factor (TF) is a major procoagulant stimulus in cancer-associated thrombosis, and tumor cell TF expression is correlated with tumor progression in several experimental tumor models (reviewed in Schaffner and Ruf 3 ). Although thrombin generated in the course of coagulation supports hematogenous metastasis 4 and regulates angiogenesis and the tumor microenvironment, 5 several lines of pharmacological and genetic evidence show that direct signaling of TF plays a pivotal role in cancer progression and angiogenesis.The TF-VIIa complex cleaves the G protein-coupled protease activated receptor (PAR) 2 6 and triggers breast cancer cells to produce a diverse repertoire of angiogenic regulators and immunemodulatory cytokines. [7][8][9] The cleavage of PAR2 activates G␣q, G␣12/13, and G␣i, and termination of signaling occurs upon internalization of the receptor that is initiated by the binding of ␤-arrestins to phosphorylated residues on the C-terminus of G protein-coupled receptors. ␤-arrestins also promote G-protein independent signaling by serving as a scaffold for activating the extracellular regulated kinases (ERK) pathway. 10 The coupling of ␤-arrestin to PAR2 results in the dephosphorylation of cofilin, which leads to the severing of actin filaments. 11 Thus, the activation of the noncanonical pathway through ␤-arrestin promotes breast cancer motility. [11][12][13][14] The TF-VIIa-signaling complex is associated with integrins and regulates ␣3␤1-dependent migration in a crosstalk that involves the TF cytoplasmic domain in noncancerous epithelial cells. 15 In cancer cells, TF is constitutively associated with ␤1 integrins that regulate TF-VIIa-PAR2-mediated induction of proangiogenic chemokines. 16 Importantly, a unique monoclonal antibody to human TF without significant anticoagulant properties specifically disrupts the interaction of TF with integrins, TF-VIIa-PAR2 cell signaling, and the growth of human breast cancer xenografts in mice, 16 demonstrating that TF-PAR2 signaling is a potential therapeutic target for cancer therapy.Although breast cancer cell PAR1 signaling is deregulated to increase invasiveness 17,18 and is partially overlapping with TF-PAR2 signaling in promoting transcriptional responses, 8 only PAR2 deficiency significantly delays the progression from adenoma to adenocarcinoma in the polyoma middle T (PyMT) model of spontaneous breast cancer development. 19 In this mouse model, the oncogenic middle T-antigen protein is expressed under a mammary-specific promoter, and mice expressing the transgene develop tumors in all mammary glands. Tumor progression resembles the human disease 20 at the molecular level (eg, downregulation of the estrogen and progesterone receptors) and, due to the relatively slow development, allows the study of complex interactions between the host and tumor cells. Because postnatal, hypoxia-induced and transplanted tumor angiogenesis is unaffected in...

Summary. Background: Factor VIIa (FVIIa) binding to tissue factor (TF) induces cell signaling via the protease activity of FVIIa and protease-activated receptor 2 (PAR2). Objective: We examined how the gene-expression profile induced by FVIIa corresponds to the profiles induced by protease-activated receptor 1 (PAR1) or PAR2 agonists using MDA-MB-231 breast carcinoma cells that constitutively express TF, PAR1 and PAR2. Results and conclusions: Out of 8500 genes, FVIIa stimulation induced differential regulation of 39 genes most of which were not previously recognized as FVIIa regulated. All genes regulated by FVIIa were similarly regulated by a PAR2 agonist peptide confirming FVIIa signaling via PAR2. An appreciable fraction of the PAR2-regulated genes was also regulated by a PAR1 agonist peptide suggesting extensive redundancy between FVIIa/PAR2 signaling and thrombin/ PAR1 signaling. The FVIIa regulated genes encode cytokines, chemokines and growth factors, and the gene repertoire induced by FVIIa in MDA-MB-231 cells is consistent with a role for TF-FVIIa signaling in regulation of a wound healing type of response. Interestingly, a number of genes regulated exclusively by FVIIa/PAR2-mediated cell signaling in MDA-MB-231 cells were regulated by thrombin and a PAR1 agonist, but not by FVIIa, in the TF-expressing glioblastoma U373 cell line.