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• W2092097766 abstract "Signal transduction induced by activated factor VII (FVIIa) was studied with baby hamster kidney (BHK) cells transfected with human tissue factor (TF). FVIIa induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) in cells expressing TF, BHK(+TF), but not in wild-type BHK(-TF) cells. BHK(+TF) cells responded to FVIIa in a dose-dependent manner, with detectable phosphorylation above 10–20 nm FVIIa. BHK cells transfected with a cytoplasmic domain-deleted version of TF, (des248–263)TF, or a C245S substitution variant of TF also supported FVIIa-induced MAPK activation. Experiments with active site-inhibited FVIIa, thrombin, factor Xa, and hirudin confirmed that the catalytic activity of FVIIa was mandatory for p44/42 MAPK activation. Furthermore, a high concentration of FVIIa in complex with soluble TF induced p44/42 MAPK phosphorylation in BHK(-TF) cells. These data suggest that TF was not directly involved in FVIIa-induced p44/42 MAPK phosphorylation but rather served to localize the action of FVIIa to the cell surface, potentially to cleave a cell surface receptor. Desensitization experiments with sequential addition of proteases suggested that the p44/42 MAPK response induced by FVIIa was distinctly different from the thrombin response, possibly involving a novel member of the protease-activated receptor family. Signal transduction induced by activated factor VII (FVIIa) was studied with baby hamster kidney (BHK) cells transfected with human tissue factor (TF). FVIIa induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) in cells expressing TF, BHK(+TF), but not in wild-type BHK(-TF) cells. BHK(+TF) cells responded to FVIIa in a dose-dependent manner, with detectable phosphorylation above 10–20 nm FVIIa. BHK cells transfected with a cytoplasmic domain-deleted version of TF, (des248–263)TF, or a C245S substitution variant of TF also supported FVIIa-induced MAPK activation. Experiments with active site-inhibited FVIIa, thrombin, factor Xa, and hirudin confirmed that the catalytic activity of FVIIa was mandatory for p44/42 MAPK activation. Furthermore, a high concentration of FVIIa in complex with soluble TF induced p44/42 MAPK phosphorylation in BHK(-TF) cells. These data suggest that TF was not directly involved in FVIIa-induced p44/42 MAPK phosphorylation but rather served to localize the action of FVIIa to the cell surface, potentially to cleave a cell surface receptor. Desensitization experiments with sequential addition of proteases suggested that the p44/42 MAPK response induced by FVIIa was distinctly different from the thrombin response, possibly involving a novel member of the protease-activated receptor family. The extrinsic pathway of blood coagulation is initiated when factor VIIa (FVIIa) 1The abbreviations used are: FVIIa, activated FVII; TF, tissue factor; sTF, TF(1-219); TFΔcyto, TF(1-247); FFR-FVIIa, FVIIa blocked in the active site withd-Phe-l-Phe-l-Arg-chloromethyl ketone; FX, factor X; MAPK, mitogen-activated protein kinase; PAR, protease-activated receptor; FCS, fetal calf serum; BHK, baby hamster kidney1The abbreviations used are: FVIIa, activated FVII; TF, tissue factor; sTF, TF(1-219); TFΔcyto, TF(1-247); FFR-FVIIa, FVIIa blocked in the active site withd-Phe-l-Phe-l-Arg-chloromethyl ketone; FX, factor X; MAPK, mitogen-activated protein kinase; PAR, protease-activated receptor; FCS, fetal calf serum; BHK, baby hamster kidneycirculating in plasma binds to the integral membrane protein, tissue factor (TF), exposed to the blood upon injury of the vessel wall. TF consists of a 219-residue (1–219) extracellular domain, a 23-residue (220–242) transmembrane domain, and a 21-residue (243–263) cytoplasmic domain. The extracellular part of TF is structured in two fibronectin type III-like domains, which shows structural and sequence homology to the cytokine receptor superfamily. The important role of TF in hemostasis and thrombotic disorders such as atherogenesis is well established (see (1Camerer E. Kolsto A.-B. Prydz H. Thromb. Res. 1996; 81: 1-41Abstract Full Text PDF PubMed Scopus (394) Google Scholar, 2Petersen L.C. Valentin S. Hedner U. Thromb. Res. 1995; 79: 1-47Abstract Full Text PDF PubMed Scopus (84) Google Scholar, 3Rapaport S. Rao M.V.L. Thromb. Haemostasis. 1995; 74: 7-17Crossref PubMed Scopus (413) Google Scholar, 4Semeraro N. Colucci M. Thromb. Haemostasis. 1997; 78: 759-764Crossref PubMed Scopus (173) Google Scholar, 5Taubman M.B. Fallon J.T. Schecter A.D. Giesen P. Mendlowitz M. Fyfe B.S. Marmur J.D. Nemerson Y. Thromb. Haemostasis. 1997; 78: 200-204Crossref PubMed Scopus (191) Google Scholar)). Recent findings suggest that TF may participate in biological processes other than hemostasis such as angiogenesis (6Shoji M. Hancock W.W. Abe K. Micko C. Casper K.A. Baine R.M. Wilcox J.N. Danave I. Dillehay D.L. Matthews E. Contrino J. Morrissey J.H. Gordon S. Edgington T.S. Kudryk B. Kreutzer D.L. Rickles F.R. Am. J. Pathol. 1998; 152: 399-411PubMed Google Scholar), embryo vascularization (7Carmeliet P. Mackman N. Moons L. Luther T. Gressens P. Van Vlaenderen I. Demunck H. Kasper M. Breier G. Evrard P. Muller M. Risau W. Edgington T. Collen D. Nature. 1996; 383: 73-75Crossref PubMed Scopus (574) Google Scholar), and tumor metastasis (8Bromberg M.E. Konigsberg W.H. Madison J.F. Pawshe A. Garen A. Proc. Natl. Acad. Sci. U. S. A. 1995; 97: 8205-8209Crossref Scopus (282) Google Scholar,9Mueller B.M. Ruf W. J. Clin. Invest. 1997; 101: 1372-1378Crossref Google Scholar). Furthermore, it has been reported that binding of FVIIa to cell surface TF-induced intracellular Ca2+ oscillations in a number of TF-expressing cells (10Rottingen J.-A. Enden T. Camerer E. Iversen J.-G. Prydz H. J. Biol. Chem. 1995; 270: 4650-4660Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 11Camerer E. Rottingen J.-A. Iversen J.-G. Prydz H. J. Biol. Chem. 1996; 271: 29034-29042Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), transient phosphorylation of tyrosine in monocytes (12Masuda M. Nakamura S. Murakami S. Komiyama Y. Takahashi H. Eur. J. Immunol. 1996; 26: 2529-2532Crossref PubMed Scopus (40) Google Scholar), alteration in gene expression in fibroblasts (13Pendurthi U.R. Alok D. Rao L.V.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12598-12603Crossref PubMed Scopus (92) Google Scholar), and enhanced expression of urokinase receptor in pancreatic cancer cells (14Taniguchi T. Kakkar A.K. Tuddenham E.G.D. Williamson R.C.N. Lemoine N.R. Cancer Res. 1998; 58: 4461-4467PubMed Google Scholar). Additional information about FVIIa/TF-induced signal transduction comes from our previous report showing that binding of FVIIa to cell surface TF resulted in phosphorylation of p44/42 MAPK (15Poulsen L.K. Jacobsen N. Sørensen B.B. Bergenhem N.C.H. Kelly J.D. Foster D.C. Thastrup O. Ezban M. Petersen L.C. J. Biol. Chem. 1998; 273: 6228-6232Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar).A potential role for the TF cytoplasmic domain in signal transduction was indicated by studies showing that TF expression markedly increased the metastatic potential of melanoma cells (16Mueller B.M. Reisfeld R.A. Edgington T.S. Ruf W. Proc. Natl. Acad. Sci U. S. A. 1992; 98: 11832-11836Crossref Scopus (337) Google Scholar) and that the pro-metastatic property was critically dependent on this domain (8Bromberg M.E. Konigsberg W.H. Madison J.F. Pawshe A. Garen A. Proc. Natl. Acad. Sci. U. S. A. 1995; 97: 8205-8209Crossref Scopus (282) Google Scholar, 9Mueller B.M. Ruf W. J. Clin. Invest. 1997; 101: 1372-1378Crossref Google Scholar). This supposition was further substantiated by experiments showing that the cytoplasmic domain of TF can be phosphorylated by a protein kinase C-dependent mechanism (17Zinocheck T.F. Roy S. Vehar G.A. J. Biol. Chem. 1992; 267: 3561-3564Abstract Full Text PDF PubMed Google Scholar) and that a synthetic peptide based on the cytoplasmic domain can work as a substrate for cell lysate protein kinase activity (18Mody R.S. Carson S.D. Biochemistry. 1997; 36: 7869-7875Crossref PubMed Scopus (41) Google Scholar). Furthermore, cysteine 245 in the TF cytoplasmic domain was shown to be acylated with long chain fatty acids (19Bach R. Konigsberg W.H. Nemerson Y. Biochemistry. 1988; 27: 4227-4231Crossref PubMed Scopus (94) Google Scholar).Although a number of studies, as referred above, suggest that TF is involved in induction of an intracellular activity, it is not clear how signal transduction is mediated across the membrane as a result of binding of FVIIa to TF, just as the role of the cytoplasmic domain of TF in this process is still unclear. The present study explores the effect of removing the TF cytoplasmic domain on p44/42 MAPK signaling and further examines the importance of FVIIa catalytic activity in mediating the activation of the p44/42 MAPK pathway. Our data show that the cytoplasmic TF domain with its putative sites for regulatory modifications is not required for FVIIa-induced p44/42 MAPK phosphorylation. Furthermore, the data provide evidence that specific FVIIa catalytic activity is required and that other serine proteases fail to mimic the response, suggesting that TF/FVIIa may be activating a novel receptor on the cell surface.DISCUSSIONThe proteolytic activity of FVIIa and the interaction with TF on the cell surface is of primary importance for initiation of blood coagulation. A number of recent studies suggest that TF may also be involved in angiogenesis, cell migration, and embryonic development (7Carmeliet P. Mackman N. Moons L. Luther T. Gressens P. Van Vlaenderen I. Demunck H. Kasper M. Breier G. Evrard P. Muller M. Risau W. Edgington T. Collen D. Nature. 1996; 383: 73-75Crossref PubMed Scopus (574) Google Scholar) and in tumor metastasis (8Bromberg M.E. Konigsberg W.H. Madison J.F. Pawshe A. Garen A. Proc. Natl. Acad. Sci. U. S. A. 1995; 97: 8205-8209Crossref Scopus (282) Google Scholar, 9Mueller B.M. Ruf W. J. Clin. Invest. 1997; 101: 1372-1378Crossref Google Scholar, 16Mueller B.M. Reisfeld R.A. Edgington T.S. Ruf W. Proc. Natl. Acad. Sci U. S. A. 1992; 98: 11832-11836Crossref Scopus (337) Google Scholar). Additional evidence for the importance of TF in biological functions other than coagulation comes from a recent observation suggesting that TF may act as a true receptor (10Rottingen J.-A. Enden T. Camerer E. Iversen J.-G. Prydz H. J. Biol. Chem. 1995; 270: 4650-4660Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 11Camerer E. Rottingen J.-A. Iversen J.-G. Prydz H. J. Biol. Chem. 1996; 271: 29034-29042Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 12Masuda M. Nakamura S. Murakami S. Komiyama Y. Takahashi H. Eur. J. Immunol. 1996; 26: 2529-2532Crossref PubMed Scopus (40) Google Scholar, 13Pendurthi U.R. Alok D. Rao L.V.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12598-12603Crossref PubMed Scopus (92) Google Scholar, 14Taniguchi T. Kakkar A.K. Tuddenham E.G.D. Williamson R.C.N. Lemoine N.R. Cancer Res. 1998; 58: 4461-4467PubMed Google Scholar, 15Poulsen L.K. Jacobsen N. Sørensen B.B. Bergenhem N.C.H. Kelly J.D. Foster D.C. Thastrup O. Ezban M. Petersen L.C. J. Biol. Chem. 1998; 273: 6228-6232Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Although it was shown that a TF-dependent intracellular activity was induced by binding of FVIIa to TF on the cell surface, it was not clear how signal transduction across the plasma membrane was accomplished. We have shown recently (15Poulsen L.K. Jacobsen N. Sørensen B.B. Bergenhem N.C.H. Kelly J.D. Foster D.C. Thastrup O. Ezban M. Petersen L.C. J. Biol. Chem. 1998; 273: 6228-6232Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) that FVIIa/TF-induced signal transduction leads to p44/42 MAPK phosphorylation. The data of the present study (Fig. 1) show that p44/42 MAPK activation is FVIIa dose-dependent and detectable at physiological levels of FVIIa. The present study confirms that the catalytic activity of FVIIa is required for FVIIa/TF-mediated p44/42 MAPK phosphorylation and also provides further evidence that down-stream coagulation factors FXa and thrombin, which might have been generated as a result of FVIIa activity, are not involved in FVIIa/TF-mediated MAPK activation.Our studies with TF-transfected BHK cells (Fig. 2) clearly demonstrated that TF mutants lacking the cytoplasmic domain or the acylation site at Cys-245 were fully capable of mediating FVIIa-induced p44/42 MAPK phosphorylation. These data exclude a mandatory role for the cytoplasmic tail TF or its acylation in FVIIa/TF-induced signaling via the MAPK pathway, although we cannot completely exclude the involvement of the tail in modulation of the response. It has been speculated that TF, because of its sequence homology to the cytokine receptors, might be capable of mediating transmembrane signal transduction by receptor dimerization or oligomerization followed by reciprocal phosphorylation of intracellular domains (35Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6934-6938Crossref PubMed Scopus (1869) Google Scholar). However, our present finding that FVIIa can induce MAP kinase activation in cells expressing truncated TF with no intracellular domain excludes such a mechanism, at least for TF/VIIa-induced MAP kinase activation. Recent preliminary observations showing that human TF protein lacking the cytoplasmic domain rescues TF−/− mouse embryos (36Parry G.C.N. Erlich J. Mackman N. Circulation. 1998; 98 Suppl. I (abstr.): 40Google Scholar) provide an indirect support to our present conclusion that the TF extracellular domain can function independently of the cytoplasmic domain in mediating signal transduction. However, one should note that our data do not necessarily rule out a possible involvement of the cytoplasmic tail in other TF-mediated signal transduction events. A requirement for the cytoplasmic domain of TF is suggested from studies on its prometastatic activity (8Bromberg M.E. Konigsberg W.H. Madison J.F. Pawshe A. Garen A. Proc. Natl. Acad. Sci. U. S. A. 1995; 97: 8205-8209Crossref Scopus (282) Google Scholar, 9Mueller B.M. Ruf W. J. Clin. Invest. 1997; 101: 1372-1378Crossref Google Scholar). Furthermore, the cytoplasmic domain of TF was shown to interact with actin-binding protein 280 (ABP-280), suggesting a role for TF in reactions associated with rearrangement of cellular cytoskeleton elements (37Ott I. Fischer E.G. Miyagi Y. Mueller B.M. Ruf W. J. Cell Biol. 1998; 140: 1241-1253Crossref PubMed Scopus (274) Google Scholar). The exact nature and the importance of these interactions in relation to the p44/42 MAPK pathway remains to be fully unraveled.The results obtained upon treatment of BHK(-TF) cells with exogenous sTF·FVIIa complex (Fig. 3) are consistent with a pertinent role for membrane-bound TF in FVIIa-induced signal transduction because of its virtue of creating a localized area of high FVIIa activity on the plasma membrane. The importance of membrane localization was further substantiated by experiments with a truncated version of FVIIa, des(1–44)FVIIa, which lacks its membrane binding γ-carboxyglutamic acid-containing domain but retains TF-binding and enhanced proteolytic activity. An exogenous complex between this FVIIa derivative and TF, sTF·des(1–44)FVIIa, induced a decreased response compared with sTF·FVIIa when added to BHK(-TF) cells (results not shown) presumably because of the diminished membrane association of this complex because of the absence of the FVIIa-membrane binding domain.The fact that FVIIa proteolytic activity is required for FVIIa-induced p44/42 MAPK phosphorylation may suggest that signal transduction is mediated by a so-called PAR, which is cleaved when FVIIa binds to TF on the cell surface. The presently known PARs (PAR 1–4) belong to a subgroup of the G protein-coupled receptor family. The thrombin receptor (PAR-1), originally cloned from a human megakaryoblastic cell line (25Vu T.K.H. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2650) Google Scholar), is best known and serves as a prototype for protease-activated receptors. The PARs are activated by a mechanism in which a protease recognizes and cleaves an extracellular N-terminal exodomain of the receptor to reveal a new N terminus containing a receptor-activating sequence that serves as a tethered ligand. Intracellular calcium mobilization and inositol phosphate formation are the most commonly reported consequences of PAR activation; however, p44/42 MAPK phosphorylation as a result of protease-catalyzed cleavage has been observed with both PAR-1 (34Pages G. Lenormand P. L'Allemain G. Chambard J.-C. Méloche S. Pousségur J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8319-8323Crossref PubMed Scopus (923) Google Scholar) and PAR-2 (31Belham C.M. Tate R.J. Scott P.H. Pemberton A.D. Miller H.R.P. Wadsworth R.M. Gould G.W. Plevin R. Biochem. J. 1996; 320: 939-946Crossref PubMed Scopus (96) Google Scholar). The intracellular reactions following activation of PAR-3 and -4 are not well characterized. The intracellular effects reported, MAPK phosphorylation (15Poulsen L.K. Jacobsen N. Sørensen B.B. Bergenhem N.C.H. Kelly J.D. Foster D.C. Thastrup O. Ezban M. Petersen L.C. J. Biol. Chem. 1998; 273: 6228-6232Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) and calcium mobilization (10Rottingen J.-A. Enden T. Camerer E. Iversen J.-G. Prydz H. J. Biol. Chem. 1995; 270: 4650-4660Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), as a result of FVIIa/TF activity are therefore fully consistent with the possible activation of a known or a hereto undiscovered member of the PAR family.Although a certain protease preference for specific PARs exists, it is well recognized that a given PAR may be cleaved by several proteases and that a given protease may cleave several PARs. This cross-reactivity complicates a functional classification of specific PARs. However, identification clues may be obtained from experiments in which receptor activation by a protease makes this receptor refractory to a second stimulus. We explored this possibility by exposing the cells to FVIIa, thrombin, or trypsin followed by a subsequent exposure to FVIIa. Pre-exposure to FVIIa and not to thrombin resulted in desensitization of the FVIIa-induced response. Exposing the cells to trypsin resulted in partial desensitization of the FVIIa-induced response. These observations indicate that receptors activated/desensitized by thrombin and probably by trypsin were not involved in FVIIa-induced signal transduction. Thrombin activates and is also likely to desensitize PAR-1, -3, and -4 but not PAR-2 (27Nystedt S. Emilsson K. Wahlestedt C. Sundelin J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9208-9212Crossref PubMed Scopus (826) Google Scholar). Thus the inability of thrombin pretreatment to abolish FVIIa-induced MAP kinase activation can be taken as evidence against the involvement of PAR-1, -3, and -4. Localization of thrombin to the cell membrane (e.g. by binding to PAR-1) might be a possible prerequisite for its desensitization of PARs 3 and 4, in which case a positive identification of PAR-1 on BHK(+TF) cells would be needed to draw conclusions about the putative role of PAR-3 or PAR-4 in TF/VIIa-induced signal transduction from our desensitization experiments. However, the observation that BHK cells respond to the PAR-1 agonist, SFLLRN, and thrombin with mobilization of intracellular Ca2+ stores 2L. C. Petersen, O. Thastrun, G. Hagel, B. B. Sørensen, P.-O. Freskgård, L. V. M. Rao, and M. Ezban, unpublished data. suggests that they do indeed express PAR-1 and may not require an additional binding site on the membrane for thrombin desensitization of PAR-3 and PAR-4. The data in Fig. 5 show that trypsin induced a significant p44/42 MAPK activation. The response had not completely returned to the basal level in 40 min, and this may have complicated the desensitization experiment. Nonetheless, FVIIa treatment did enhance MAP kinase activation in trypsin-pretreated cells. Furthermore, treatment of the cells with 300 μm of the murine PAR-2 agonist, SLIGRL, did not induce p44/42 MAPK activation (results not shown) as was expected from PAR-2 activation experiments with rat aortic smooth muscle cells and bovine fibroblasts (31Belham C.M. Tate R.J. Scott P.H. Pemberton A.D. Miller H.R.P. Wadsworth R.M. Gould G.W. Plevin R. Biochem. J. 1996; 320: 939-946Crossref PubMed Scopus (96) Google Scholar). This raises the possibility that BHK cells may not express PAR-2 and also makes it unlikely that PAR-2 is involved in FVIIa-induced signal transduction. However, the observation of a partly desensitized FVIIa response could indicate that trypsin may, in addition to PAR-2, also cleave the putative receptor activated by FVIIa. So far then, the combined data on characterization of FVIIa-induced signaling seems to exclude the currently known PARs as targets for FVIIa activation, making it possible that a hereto undiscovered member of the PAR family is responsible for FVIIa-induced signal transduction.The prometastatic function of TF-expressing cells appears to depend on the concerted action of FVIIa/TF activity and certain reactions involving the cytoplasmic domain of TF (8Bromberg M.E. Konigsberg W.H. Madison J.F. Pawshe A. Garen A. Proc. Natl. Acad. Sci. U. S. A. 1995; 97: 8205-8209Crossref Scopus (282) Google Scholar). This might suggest the existence of a close connection between dynamic modifications (phosphorylation or acylation) of the TF cytoplasmic domain and the TF/FVIIa-induced signal transduction leading to p44/42 MAPK phosphorylation. The activated p44/42 MAPK is a serine/threonine-specific protein kinase that might possibly phosphorylate one or more serine residues present in the TF cytoplasmic domain. G protein-coupled receptors are known to activate parallel signal transduction pathways e.g. activation of phospholipase C-β, phosphoinositide hydrolysis, and formation of inositol trisphosphate and diacylglycerol, leading to Ca2+mobilization and activation of protein kinase C. These reaction might well be induced by a putative PAR activation by TF-bound FVIIa and result in dynamic covalent modification of the TF tail. We are currently pursuing these possibilities. The extrinsic pathway of blood coagulation is initiated when factor VIIa (FVIIa) 1The abbreviations used are: FVIIa, activated FVII; TF, tissue factor; sTF, TF(1-219); TFΔcyto, TF(1-247); FFR-FVIIa, FVIIa blocked in the active site withd-Phe-l-Phe-l-Arg-chloromethyl ketone; FX, factor X; MAPK, mitogen-activated protein kinase; PAR, protease-activated receptor; FCS, fetal calf serum; BHK, baby hamster kidney1The abbreviations used are: FVIIa, activated FVII; TF, tissue factor; sTF, TF(1-219); TFΔcyto, TF(1-247); FFR-FVIIa, FVIIa blocked in the active site withd-Phe-l-Phe-l-Arg-chloromethyl ketone; FX, factor X; MAPK, mitogen-activated protein kinase; PAR, protease-activated receptor; FCS, fetal calf serum; BHK, baby hamster kidneycirculating in plasma binds to the integral membrane protein, tissue factor (TF), exposed to the blood upon injury of the vessel wall. TF consists of a 219-residue (1–219) extracellular domain, a 23-residue (220–242) transmembrane domain, and a 21-residue (243–263) cytoplasmic domain. The extracellular part of TF is structured in two fibronectin type III-like domains, which shows structural and sequence homology to the cytokine receptor superfamily. The important role of TF in hemostasis and thrombotic disorders such as atherogenesis is well established (see (1Camerer E. Kolsto A.-B. Prydz H. Thromb. Res. 1996; 81: 1-41Abstract Full Text PDF PubMed Scopus (394) Google Scholar, 2Petersen L.C. Valentin S. Hedner U. Thromb. Res. 1995; 79: 1-47Abstract Full Text PDF PubMed Scopus (84) Google Scholar, 3Rapaport S. Rao M.V.L. Thromb. Haemostasis. 1995; 74: 7-17Crossref PubMed Scopus (413) Google Scholar, 4Semeraro N. Colucci M. Thromb. Haemostasis. 1997; 78: 759-764Crossref PubMed Scopus (173) Google Scholar, 5Taubman M.B. Fallon J.T. Schecter A.D. Giesen P. Mendlowitz M. Fyfe B.S. Marmur J.D. Nemerson Y. Thromb. Haemostasis. 1997; 78: 200-204Crossref PubMed Scopus (191) Google Scholar)). Recent findings suggest that TF may participate in biological processes other than hemostasis such as angiogenesis (6Shoji M. Hancock W.W. Abe K. Micko C. Casper K.A. Baine R.M. Wilcox J.N. Danave I. Dillehay D.L. Matthews E. Contrino J. Morrissey J.H. Gordon S. Edgington T.S. Kudryk B. Kreutzer D.L. Rickles F.R. Am. J. Pathol. 1998; 152: 399-411PubMed Google Scholar), embryo vascularization (7Carmeliet P. Mackman N. Moons L. Luther T. Gressens P. Van Vlaenderen I. Demunck H. Kasper M. Breier G. Evrard P. Muller M. Risau W. Edgington T. Collen D. Nature. 1996; 383: 73-75Crossref PubMed Scopus (574) Google Scholar), and tumor metastasis (8Bromberg M.E. Konigsberg W.H. Madison J.F. Pawshe A. Garen A. Proc. Natl. Acad. Sci. U. S. A. 1995; 97: 8205-8209Crossref Scopus (282) Google Scholar,9Mueller B.M. Ruf W. J. Clin. Invest. 1997; 101: 1372-1378Crossref Google Scholar). Furthermore, it has been reported that binding of FVIIa to cell surface TF-induced intracellular Ca2+ oscillations in a number of TF-expressing cells (10Rottingen J.-A. Enden T. Camerer E. Iversen J.-G. Prydz H. J. Biol. Chem. 1995; 270: 4650-4660Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 11Camerer E. Rottingen J.-A. Iversen J.-G. Prydz H. J. Biol. Chem. 1996; 271: 29034-29042Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), transient phosphorylation of tyrosine in monocytes (12Masuda M. Nakamura S. Murakami S. Komiyama Y. Takahashi H. Eur. J. Immunol. 1996; 26: 2529-2532Crossref PubMed Scopus (40) Google Scholar), alteration in gene expression in fibroblasts (13Pendurthi U.R. Alok D. Rao L.V.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12598-12603Crossref PubMed Scopus (92) Google Scholar), and enhanced expression of urokinase receptor in pancreatic cancer cells (14Taniguchi T. Kakkar A.K. Tuddenham E.G.D. Williamson R.C.N. Lemoine N.R. Cancer Res. 1998; 58: 4461-4467PubMed Google Scholar). Additional information about FVIIa/TF-induced signal transduction comes from our previous report showing that binding of FVIIa to cell surface TF resulted in phosphorylation of p44/42 MAPK (15Poulsen L.K. Jacobsen N. Sørensen B.B. Bergenhem N.C.H. Kelly J.D. Foster D.C. Thastrup O. Ezban M. Petersen L.C. J. Biol. Chem. 1998; 273: 6228-6232Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). A potential role for the TF cytoplasmic domain in signal transduction was indicated by studies showing that TF expression markedly increased the metastatic potential of melanoma cells (16Mueller B.M. Reisfeld R.A. Edgington T.S. Ruf W. Proc. Natl. Acad. Sci U. S. A. 1992; 98: 11832-11836Crossref Scopus (337) Google Scholar) and that the pro-metastatic property was critically dependent on this domain (8Bromberg M.E. Konigsberg W.H. Madison J.F. Pawshe A. Garen A. Proc. Natl. Acad. Sci. U. S. A. 1995; 97: 8205-8209Crossref Scopus (282) Google Scholar, 9Mueller B.M. Ruf W. J. Clin. Invest. 1997; 101: 1372-1378Crossref Google Scholar). This supposition was further substantiated by experiments showing that the cytoplasmic domain of TF can be phosphorylated by a protein kinase C-dependent mechanism (17Zinocheck T.F. Roy S. Vehar G.A. J. Biol. Chem. 1992; 267: 3561-3564Abstract Full Text PDF PubMed Google Scholar) and that a synthetic peptide based on the cytoplasmic domain can work as a substrate for cell lysate protein kinase activity (18Mody R.S. Carson S.D. Biochemistry. 1997; 36: 7869-7875Crossref PubMed Scopus (41) Google Scholar). Furthermore, cysteine 245 in the TF cytoplasmic domain was shown to be acylated with long chain fatty acids (19Bach R. Konigsberg W.H. Nemerson Y. Biochemistry. 1988; 27: 4227-4231Crossref PubMed Scopus (94) Google Scholar). Although a number of studies, as referred above, suggest that TF is involved in induction of an intracellular activity, it is not clear how signal transduction is mediated across the membrane as a result of binding of FVIIa to TF, just as the role of the cytoplasmic domain of TF in this process is still unclear. The present study explores the effect of removing the TF cytoplasmic domain on p44/42 MAPK signaling and further examines the importance of FVIIa catalytic activity in mediating the activation of the p44/42 MAPK pathway. Our data show that the cytoplasmic TF domain with its putative sites for regulatory modifications is not required for FVIIa-induced p44/42 MAPK phosphorylation. Furthermore, the data provide evidence that specific FVIIa catalytic activity is required and that other serine proteases fail to mimic the response, suggesting that TF/FVIIa may be activating a novel receptor on the cell surface. DISCUSSIONThe proteolytic activity of FVIIa and the interaction with TF on the cell surface is of primary importance for initiation of blood coagulation. A number of recent studies suggest that TF may also be involved in angiogenesis, cell migration, and embryonic development (7Carmeliet P. Mackman N. Moons L. Luther T. Gressens P. Van Vlaenderen I. Demunck H. Kasper M. Breier G. Evrard P. Muller M. Risau W. Edgington T. Collen D. Nature. 1996; 383: 73-75Crossref PubMed Scopus (574) Google Scholar) and in tumor metastasis (8Bromberg M.E. Konigsberg W.H. Madison J.F. Pawshe A. Garen A. Proc. Natl. Acad. Sci. U. S. A. 1995; 97: 8205-8209Crossref Scopus (282) Google Scholar, 9Mueller B.M. Ruf W. J. Clin. Invest. 1997; 101: 1372-1378Crossref Google Scholar, 16Mueller B.M. Reisfeld R.A. Edgington T.S. Ruf W. Proc. Natl. Acad. Sci U. S. A. 1992; 98: 11832-11836Crossref Scopus (337) Google Scholar). Additional evidence for the importance of TF in biological functions other than coagulati" @default.
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