Tissue Factor Signaling (1999)

Cell Biology of Tissue Factor Transmembrane receptor tissue factor (TF) initiates the coagulation pathways by serving as the cofactor for coagulation factor VIIa.1 The extracellular domain of TF consists of two fibronectin-type III modules in the orientation of a typical class II cytokine receptor extracellular domain,2 confirming the earlier classification of TF as a member of the cytokine receptor family.3 The evolutionary origin of TF has elicited broad interest in the question of whether this cell surface receptor has retained the capacity for signal transduction, the primary function of cytokine receptors. However, the TF cytoplasmic domain is only 21 amino acid residues in length and lacks close similarity with the cytokine receptor membrane-proximal motifs that mediate binding of the non-receptor Janus kinases (Jaks), which are essential for cytokine receptor signaling.4 These structural characteristics argue that TF signaling probably does not follow the rules of typical cytokine receptor signal transduction. An association of the TF cytoplasmic domain with intracellular signaling pathways is suggested from studies that analyze the phosphorylation of Ser/Thr residues in the TF tail. Phorbolester stimulation of TF-transfected human kidney cells results in serine phosphorylation of the TF cytoplasmic domain.5 In comparing the TF cytoplasmic domains of various species, two blocks of sequence conservation are apparent (Fig. 1). First, a cluster of basic residues is found proximal to the membrane, likely serving an optimal membrane anchor. Two serine residues in human TF, Ser253 and Ser258, are further imbedded in a block of conserved residues, and a Ser or Thr residues at position 258 is invariably followed by Pro in all species. A synthetic peptide corresponding to residue 245-263 of the TF cytoplasmic domain has recently been shown to be a substrate for an as yet to be identified protein kinase in an in vitro assay.6 Phosphorylation occurs on either three or two positions, the latter likely being Ser253 and Ser258. 6 The regulation of TF cytoplasmic domain phosphorylation in cells is presently poorly characterized. Post-translational modification of the TF cytoplasmic domain may depend on the cellular localization of TF, or alternatively, may direct and localize TF to specific cell surface microdomains. TF forms homodimers on cultered cells,7 and stimulation of cells with the Ca2+ ionophore ionomycin dissociates TF homodimers.8 It is unclear whether the dimeric or monomeric forms of TF preferentially localize to certain areas of the cell surface. On cultured cells, TF is found on the smooth areas of the plasma membrane, 9 in small plasmalemma vesicles, termed caveolae,10,11 and on cellular processes10,12 in association with cytoskeletal structures. 12-14 The localization of TF to these different membrane domains is regulated by extracellular interactions of TF. For example, binding of the TF-factor VIIa complex to its cell-associated TF pathway inhibitor, (TFPI) in the presence of factor Xa translocates the quaternary complex into caveolae and membrane domains that are highly enriched in glycosphingolipids.10 In this lipid environment, unlike the anionic parts of the membrane, the proteolytic function of TF is negligible, suggesting that cell surface trafficking of TF is a major mechanism to regulate procoagulant function. Glycosphingolipid-rich membranes and caveolae are also cellular sites that actively participate in the regulation of signaling pathways,15 and phosphorylation of the TF cytoplasmic domain may occur following the cellular trafficking of TF into these microdomains of the cell membrane. Signaling Upon Factor VIIa Binding to Tissue Factor A number of publications have described cell signaling that involves TF (Table 1). Several of these studies analyze the effect of binding of the physiological ligand factor VIIa to TF-expressing cells. Specificity controls clearly demonstrate that these signaling events are dependent on TF. For example, factor VIIa-induced Ca2+ transients in endothelial cells only occur when TF is upregulated by cytokine stimulation.16 Cells that do not express TF acquire the capability to respond to factor VIIa by Ca2+ flux16 or gene expression,17 when transfected with an expression construct encoding for TF. Third, inhibitory antibodies to TF block the factor VIIa-induced responses in TF-expressing cells.16-21 Do these data provide evidence that binding of ligand factor VIIa to TF is sufficient to induce the signaling events, i.e. that TF functions as a signal transducer? Several studies have addressed this issue by the use of active site blocked factor VIIa that binds with high affinity to TF.22 Catalytically inactive factor VIIa fails to induce the signaling responses ranging from Ca2+ fluxes23 to mitogen-activated protein (MAP) kinase activation17 and gene induction.17,19,20 The TF-factor VIIa complex is chemotactic for smooth muscle cells, and chemotaxis is abolished upon active site modification of factor VIIa.24 Taken together, these studies strongly suggest that a signaling cascade involving the proteolytic function of factor VIIa can be initiated upon binding of factor VIIa to TF (Fig. 2). It is important to consider whether these effects of factor VIIa are mediated through downstream activation of traces of culture medium-derived coagulation factors that stay associated with the cells under the experimental conditions. This possibility has been addressed in several studies by the use of specific inhibitors of factor Xa17 or thrombin.19,23,24 These inhibitors are ineffective in blocking the cellular responses to factor VIIa, demonstrating that factor VIIa does not signal through the initiation of the coagulation cascade and the indirect triggering of the known protease-activated thrombin receptors.