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...

Abstract. The ability to regulate proteolytic functions is critical to cell biology. We describe eveiats that regulate the initiation of the coagulation cascade on endothelial cell surfaces. The transmembrane protease receptor tissue factor (TF) triggers coagulation by forming an enzymatic complex with the serine protease factor VIIa (VIIa) that activates substrate factor X to the protease factor Xa (Xa). Feedback inhibition of the TF.VIIa enzymatic complex is achieved by the formation of a quaternary complex of TF.VIIa, Xa, and the Kunitz-type inhibitor tissue factor pathway inhibitor (TFPI). Concomitant with the downregulation of TF.VIIa function on endothelial cells, we demonstrate by immunogold EM that TF redistributes to caveolae. Consistently, TF translocates from the Triton X-100-soluble membrane fractions to low-density, detergentinsoluble microdomains that inefficiently support TF.VIIa proteolytic function. Downregulation of TF.VIIa function is dependent on quaternary complex formation with TFPI that is detected predominantly in detergent-insoluble microdomains. Partitioning of TFPI into low-density fractions results from the association of the inhibitor with glycosyl phosphatidylinositol-anchored binding sites on external membranes. Free Xa is not efficiently bound by cell-associated TFPI; hence, we propose that the transient ternary complex of TF.VIIa with Xa supports translocation and assembly with TFPI in glycosphingolipid-rich microdomains. The redistribution of TF provides evidence for an assembly-dependent translocation of the inhibited TF initiation complex into caveolae, thus implicating caveolae in the regulation of cell surface proteolytic activity.C ELLS accomplish tasks of invasion, migration, tissue remodeling and cell-cell communication in part through the regulated expression of cell-associated protease systems. Cell surface protease cascades are triggered by the upregulation of protease receptors, such as the urokinase receptor of the fibrinolytic system (14) and tissue factor (TF) 1 that initiates the plasma coagulation pathways (48). Based on structural homology in the extracellular domain, TF is classified as a member of the cytokine receptor family and most closely related to the interferon and IL-10 receptors (48). However, in contrast to the four helix bundle ligands of the latter, TF binds the multidomain serine protease factor VIIa (VIIa) with subnanomolar affinity. The assembly of the protease into the TF-VIIa complex results in markedly enhanced proAddress all correspondence to Wolfram Ruf, Department of Immunology, IMM-17, The

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We determined whether human NK cells could contribute to immune defenses against Mycobacterium tuberculosis through production of IL-22. CD3−CD56+ NK cells produced IL-22 when exposed to autologous monocytes and γ-irradiated M. tuberculosis, and this depended on the presence of IL-15 and IL-23, but not IL-12 or IL-18. IL-15-stimulated NK cells expressed 10.6 times more DAP10 mRNA compared with control NK cells, and DAP10 siRNA inhibited IL-15-mediated IL-22 production by NK cells. Soluble factors produced by IL-15-activated NK cells inhibited growth of M. tuberculosis in macrophages, and this effect was reversed by anti-IL-22. Addition of rIL-22 to infected macrophages enhanced phagolysosomal fusion and reduced growth of M. tuberculosis. We conclude that NK cells can contribute to immune defenses against M. tuberculosis through production of IL-22, which inhibits intracellular mycobacterial growth by enhancing phagolysosomal fusion. IL-15 and DAP-10 elicit IL-22 production by NK cells in response to M. tuberculosis.