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...

Although factor VII/factor VIIa (FVII/FVIIa) is known to interact with many non-vascular cells, activated monocytes, and endothelial cells via its binding to tissue factor (TF), the interaction of FVII/FVIIa with unperturbed endothelium and the role of this interaction in clearing FVII/FVIIa from the circulation are unknown. To investigate this, in the present study we examined the binding of radiolabeled FVIIa to endothelial cells and its subsequent internalization. 125 I-FVIIa bound to non-stimulated human umbilical vein endothelial cells (HUVEC) in time-and dose-dependent manner. The binding is specific and independent of TF and negatively charged phospholipids. Protein C and monoclonal antibodies to endothelial cell protein C receptor (EPCR) blocked effectively 125 I-FVIIa binding to HUVEC. FVIIa binding to EPCR is confirmed by demonstrating a marked increase in 125 I-FVIIa binding to CHO cells that had been stably transfected with EPCR compared with the wild-type. Binding analysis revealed that FVII, FVIIa, protein C, and activated protein C (APC) bound to EPCR with similar affinity. FVIIa binding to EPCR failed to accelerate FVIIa activation of factor X or protease-activated receptors. FVIIa binding to EPCR was shown to facilitate FVIIa endocytosis. Pharmacological concentrations of FVIIa were found to impair partly the EPCR-dependent protein C activation and APC-mediated cell signaling. Overall, the present data provide convincing evidence that EPCR serves as a cellular binding site for FVII/FVIIa. Further studies are needed to evaluate the pathophysiological consequences and relevance of FVIIa binding to EPCR.Despite the progress that has been made in understanding the biochemistry and pathophysiology of the coagulation cascade events, the clearance mechanism of the various coagulation proteins from the circulation is still unclear. The marked differences in circulating half-lives of factor VII (FVII) 4 and FVIIa compared with those of zymogen and the enzyme forms of other vitamin K-dependent coagulation proteins (1-7) suggest that there may be a specific and distinct clearance mechanism for FVII/FVIIa. Although tissue factor (TF), the * This work was supported by Grants HL 58869 and HL 65500 from the National Institutes of Health. 4 The abbreviations used are: FVII, factor VII; FVIIa, activated factor VII; TF, tissue factor; EPCR, endothelial cell protein C receptor; APC, activated protein C; PAR, protease-activated receptor; AP, alkaline phosphatase; TFPI, tissue factor pathway inhibitor; AT, antithrombin; FACS, fluorescence-activated cell sorter; TNF, tumor necrosis factor; IL, interleukin. NIH Public Access Cell CulturePrimary human umbilical vein endothelial cells (HUVEC) were purchased from Cambrex Bio Science (Walkersville, MD). The cells were cultured to confluency at 37 °C and 5% CO 2 in a humidified incubator in EBM-2 basal media supplemented with growth supplements (Cambrex Bio Science) and 5% fetal bovine serum. Endothelial cell passages between 3 and 10 were used in the present stud...

Extracellular interactions of plasma clotting factor VIIa (FVIIa) with tissue factor (TF) on cell surfaces trigger the intracellular signaling events. At present, it is unclear how these signals influence phenotype. To elucidate this, we have used cDNA microarray technology to examine changes in transcriptional program in human fibroblasts in response to exposure to FVIIa. cDNA microarrays revealed that FVIIa binding to TF up-regulated the expression of Cyr61 and CTGF (connective tissue growth factor), the genes that encode extracellular matrix signaling proteins Cyr61 and CTGF, respectively. Northern blot analysis confirmed that FVIIa binding to TF markedly increased the expression of Cyr61 and CTGF in a time- and dose-dependent manner. FVIIa catalytic activity is required for the gene induction. In addition to FVIIa, thrombin also induced the expression of Cyr61 and CTGF. Hirudin abolished the thrombin-induced expression of these mRNAs but not the FVIIa-induced expression. FVIIa-induced expression of Cyr61 appears not to involve the currently known protease-activated receptors (PARs), whereas thrombin-induced expression involves the activation of PAR1 and possibly an additional PAR. Various intracellular signaling pathway inhibitors exhibited different inhibitory pattern on FVIIa and thrombin-induced up-regulation of Cyr61. Cyr61 and CTGF could act as downstream mediators of FVIIa x TF in affecting various biological processes.

Local derangements of fibrin turnover and plasminogen activator inhibitor (PAI)-1 have been implicated in the pathogenesis of pleural injury. However, their role in the control of pleural organization has been unclear. We found that a C57Bl/6j mouse model of carbon black/bleomycin (CBB) injury demonstrates pleural organization resulting in pleural rind formation (14 d). In transgenic mice overexpressing human PAI-1, intrapleural fibrin deposition was increased, but visceral pleural thickness, lung volumes, and compliance were comparable to wild type. CBB injury in PAI-1 2/2 mice significantly increased visceral pleural thickness (P , 0.001), elastance (P , 0.05), and total lung resistance (P , 0.05), while decreasing lung compliance (P , 0.01) and lung volumes (P , 0.05). Collagen, a-smooth muscle actin, and tissue factor were increased in the thickened visceral pleura of PAI-1 2/2 mice. Colocalization of a-smooth muscle actin and calretinin within pleural mesothelial cells was increased in CBB-injured PAI-1 2/2 mice. Thrombin, factor Xa, plasmin, and urokinase induced mesothelial-mesenchymal transition, tissue factor expression, and activity in primary human pleural mesothelial cells. In PAI-1 2/2 mice, D-dimer and thrombin-antithrombin complex concentrations were increased in pleural lavage fluids. The results demonstrate that PAI-1 regulates CBB-induced pleural injury severity via unrestricted fibrinolysis and cross-talk with coagulation proteases. Whereas overexpression of PAI-1 augments intrapleural fibrin deposition, PAI-1 deficiency promotes profibrogenic alterations of the mesothelium that exacerbate pleural organization and lung restriction.

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. Key Words: tissue factor Ⅲ factor VIIa Ⅲ protease activated receptors Ⅲ cell signaling T he 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 proteincoupled 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 VIIaTF 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, monocytes 3 and endothelial cells 4 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 ac...