Protease-activated Receptor 2-dependent Phosphorylation of the Tissue Factor Cytoplasmic Domain

Tissue factor (TF) is the physiological activator of the coagulation cascade that plays pathophysiological roles in metastasis, angiogenesis, and inflammation. Downstream in coagulation, thrombin is the central protease that signals through G protein-coupled, protease-activated receptors (PARs). However, the TF-VIIa-Xa complex upstream in coagulation also activates PAR1 and 2. Here, we address the question of whether signaling of the TF initiation complex is a relevant pathway that leads to TF cytoplasmic domain phosphorylation. In heterologous expression systems and primary endothelial cells, we demonstrate that the ternary TF-VIIa-Xa complex induces TF phosphorylation specifically by activating PAR2 but not through PAR1 signaling. In addition, TF cytoplasmic domain phosphorylation is induced only by TF-dependent signaling but not by other coagulation factors in endothelial cells. Phosphorylation of the Pro-directed kinase target site Ser258 is dependent on prior phosphorylation of Ser253 by protein kinase C (PKC) α. TF phosphorylation is somewhat delayed and coincides with sustained PKCα activation downstream of PAR2 but not PAR1 signaling. Phosphatidylcholine-dependent phospholipase C is the major pathway that leads to prolonged PKCα recruitment downstream of PAR2. Thus, PAR2 signaling specifically phosphorylates TF in a receptor cross-talk that distinguishes upstream from downstream coagulation protease signaling. Tissue factor (TF) is the physiological activator of the coagulation cascade that plays pathophysiological roles in metastasis, angiogenesis, and inflammation. Downstream in coagulation, thrombin is the central protease that signals through G protein-coupled, protease-activated receptors (PARs). However, the TF-VIIa-Xa complex upstream in coagulation also activates PAR1 and 2. Here, we address the question of whether signaling of the TF initiation complex is a relevant pathway that leads to TF cytoplasmic domain phosphorylation. In heterologous expression systems and primary endothelial cells, we demonstrate that the ternary TF-VIIa-Xa complex induces TF phosphorylation specifically by activating PAR2 but not through PAR1 signaling. In addition, TF cytoplasmic domain phosphorylation is induced only by TF-dependent signaling but not by other coagulation factors in endothelial cells. Phosphorylation of the Pro-directed kinase target site Ser258 is dependent on prior phosphorylation of Ser253 by protein kinase C (PKC) α. TF phosphorylation is somewhat delayed and coincides with sustained PKCα activation downstream of PAR2 but not PAR1 signaling. Phosphatidylcholine-dependent phospholipase C is the major pathway that leads to prolonged PKCα recruitment downstream of PAR2. Thus, PAR2 signaling specifically phosphorylates TF in a receptor cross-talk that distinguishes upstream from downstream coagulation protease signaling. Cell signaling through G protein-coupled protease-activated receptors (PARs) 1The abbreviations used are: PAR, protease-activated receptor; PKC, protein kinase C; PLC, phospholipase C; PMA, phorbol 12-myristate 13-acetate; CHO, Chinese hamster ovary; NAP, nematode anticoagulant protein; TF, tissue factor; ERK, extracellular signal-regulated kinase; TFCD, TF cytoplasmic domain; HUVEC, human umbilical vein endothelial cell(s); PI, phosphatidylinositol; PC, phosphatidylcholine; PLC, phospholipase C. is closely linked to the activation of the coagulation protease cascade (1Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2175) Google Scholar). Genetic evidence established an essential in vivo role of PAR signaling for platelet activation and the hemostatic response (2Sambrano G.R. Weiss E.J. Zheng Y.-W. Coughlin S.R. Nature. 2001; 413: 26-27Crossref PubMed Scopus (464) Google Scholar, 3Weiss E.J. Hamilton J.R. Lease K.E. Coughlin S.R. Blood. 2002; 100: 3240-3244Crossref PubMed Scopus (166) Google Scholar). However, PAR signaling also has important nonhemostatic functions in development (4Griffin C.T. Srinavasan Y. Zheng Y.-W. Huang W. Coughlin S.R. Science. 2001; 293: 1666-1670Crossref PubMed Scopus (246) Google Scholar), inflammation (5Coughlin S.R. Camerer E. J. Clin. Invest. 2003; 111: 25-27Crossref PubMed Scopus (220) Google Scholar), and tumor biology (6Yin Y.J. Salah Z. Grisaru-Granovsky S. Cohen I. Cohen Even-Ram S. Maoz M. Uziely B. Bar-Shavit R. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 940-944Crossref PubMed Scopus (76) Google Scholar). All PARs, except for PAR2, are directly activated by thrombin, and PAR signaling is typically attributed to the action of thrombin in vivo. However, in vitro evidence indicates that thrombin receptors are also targets for other proteases at meaningful physiological concentrations (7Pendurthi U.R. Ngyuen M. Andrade-Gordon P. Petersen L.C. Rao L.V.M. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 1421-1426Crossref PubMed Scopus (76) Google Scholar, 8Riewald M. Kravchenko V.V. Petrovan R.J. O'Brien P.J. Brass L.F. Ulevitch R.J. Ruf W. Blood. 2001; 97: 3109-3116Crossref PubMed Scopus (179) Google Scholar, 9Riewald M. Ruf W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7742-7747Crossref PubMed Scopus (284) Google Scholar, 10Riewald M. Petrovan R.J. Donner A. Mueller B.M. Ruf W. Science. 2002; 296: 1880-1882Crossref PubMed Scopus (755) Google Scholar). Cellular receptors can concentrate serine proteases at the cell surface for PAR cleavage (11Ruf W. Dorfleutner A. Riewald M. J. Thromb. Haemostasis. 2003; 1: 1495-1503Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). More importantly, protease co-factors, such as tissue factor (TF) and endothelial cell protein C receptor, couple the initiation of the pro- and anti-coagulant pathways to PAR signaling by retaining transiently the initial protease products Xa and activated protein C on the cell surface (9Riewald M. Ruf W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7742-7747Crossref PubMed Scopus (284) Google Scholar, 10Riewald M. Petrovan R.J. Donner A. Mueller B.M. Ruf W. Science. 2002; 296: 1880-1882Crossref PubMed Scopus (755) Google Scholar). Different PARs produce only partially redundant signaling responses in vascular cells. In the case of PAR1 and PAR2, the predominant endothelial cell expressed PARs, several immediate early gene induction events are overlapping, but only PAR1 signaling induces the mRNA for monocyte chemoattractant protein 1 (10Riewald M. Petrovan R.J. Donner A. Mueller B.M. Ruf W. Science. 2002; 296: 1880-1882Crossref PubMed Scopus (755) Google Scholar). PAR signaling induces distinct cytoskeletal effects. PAR1 is a potent activator of the small GTPase Rho, which leads to the disruption of intercellular junctions and to increased permeability of the endothelium (12Klarenbach S.W. Chipiuk A. Nelson R.C. Hollenberg M.D. Murray A.G. Circ. Res. 2003; 92: 272-278Crossref PubMed Scopus (100) Google Scholar, 13Vouret-Craviari V. Grall D. Van Obberghen-Schilling E. J. Thromb. Haemostasis. 2003; 1: 1103-1111Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In contrast, PAR2 activation promotes cell motility by activation of the Rac/p21-activated kinase pathway and by forming a scaffolding complex containing β-arrestin and extracellular signal-regulated kinase (ERK). This complex is implicated in pseudopodia extension and chemotaxis (14DeFea K.A. Zalevsky J. Thoma M.S. Déry O. Mullins R.D. Bunnett N. J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (696) Google Scholar, 15Ge L. Ly Y. Hollenberg M. DeFea K. J. Biol. Chem. 2003; 278: 34418-34426Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). PAR selectivity is corroborated by in vivo studies demonstrating that agonist peptides specific for PAR1 and PAR2 elicit selective responses in vascular tone (16Damiano B.P. Cheung W.M. Santulli R.J. Fung-Leung W.P. Ngo K. Ye R.D. Darrow A.L. Derian C.K. De Garavilla L. Andrade-Gordon P. J. Pharmacol. Exp. Ther. 1999; 288: 671-678PubMed Google Scholar, 17Hamilton J.R. Moffatt J.D. Frauman A.G. Cocks T.M. J. Cardiovasc. Pharmacol. 2001; 38: 108-119Crossref PubMed Scopus (56) Google Scholar) and that developmental early embryonic lethality is specific for genetic deletion of PAR1 (4Griffin C.T. Srinavasan Y. Zheng Y.-W. Huang W. Coughlin S.R. Science. 2001; 293: 1666-1670Crossref PubMed Scopus (246) Google Scholar). Thus, protease signaling specificity is in part dependent on the signal transducing PAR, but additional complexity possibly arises from the co-factors/protease receptors that assist PAR cleavage. In the initiation phase of coagulation, TF is involved in two signaling complexes: the TF-VIIa complex that activates PAR2 and the ternary TF-VIIa-Xa complex in which Xa cleaves PAR1 or PAR2 (8Riewald M. Kravchenko V.V. Petrovan R.J. O'Brien P.J. Brass L.F. Ulevitch R.J. Ruf W. Blood. 2001; 97: 3109-3116Crossref PubMed Scopus (179) Google Scholar, 9Riewald M. Ruf W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7742-7747Crossref PubMed Scopus (284) Google Scholar, 18Camerer E. Huang W. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5255-5260Crossref PubMed Scopus (619) Google Scholar, 19Camerer E. Kataoka H. Kahn M. Lease K. Coughlin S.R. J. Biol. Chem. 2002; 277: 16081-16087Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Because the ternary complex activates PAR1 and PAR2, it is an unresolved question whether immediate or delayed responses distinguish between TF-dependent signaling through PAR1 or PAR2. In this study we further characterize how the TF cytoplasmic domain becomes phosphorylated in endothelial cells. Although TF phosphorylation can be induced by phorbol 12-myristate 13-acetate (PMA) stimulation (20Zioncheck T.F. Roy S. Vehar G.A. J. Biol. Chem. 1992; 267: 3561-3564Abstract Full Text PDF PubMed Google Scholar, 21Mody R.S. Carson S.D. Biochemistry. 1997; 36: 7869-7875Crossref PubMed Scopus (41) Google Scholar, 22Dorfleutner A. Ruf W. Blood. 2003; 102: 3998-4005Crossref PubMed Scopus (87) Google Scholar), to date no agonist pathway of potential physiological relevance has been identified that leads to TF cytoplasmic domain phosphorylation. Here, we show that PAR2 but not PAR1 signaling induces TF cytoplasmic domain phosphorylation. PMA-induced TF phosphorylation in endothelial cells was found to involve PKCα-mediated phosphorylation of Ser253 (22Dorfleutner A. Ruf W. Blood. 2003; 102: 3998-4005Crossref PubMed Scopus (87) Google Scholar) and subsequent phosphorylation of Ser258 by a potential Pro-directed kinase (20Zioncheck T.F. Roy S. Vehar G.A. J. Biol. Chem. 1992; 267: 3561-3564Abstract Full Text PDF PubMed Google Scholar, 21Mody R.S. Carson S.D. Biochemistry. 1997; 36: 7869-7875Crossref PubMed Scopus (41) Google Scholar, 22Dorfleutner A. Ruf W. Blood. 2003; 102: 3998-4005Crossref PubMed Scopus (87) Google Scholar). Whereas PKCα is activated downstream of PAR1 and PAR2 signaling, sustained membrane recruitment of PKCα is specific for PAR2 signaling and coincides with the onset of TF cytoplasmic domain phosphorylation. TF phosphorylation is a characteristic response of the initiation phase of coagulation and thus distinguishes PAR signaling in the upstream and downstream coagulation reaction. Materials—VIIa, inactive Ser195 to Ala VIIa (iVIIa), factors IXa, X and Xa, thrombin, and PAR agonist peptides TFLLRNPNDK (PAR1) and SLIGRL (PAR2) were as described (8Riewald M. Kravchenko V.V. Petrovan R.J. O'Brien P.J. Brass L.F. Ulevitch R.J. Ruf W. Blood. 2001; 97: 3109-3116Crossref PubMed Scopus (179) Google Scholar, 9Riewald M. Ruf W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7742-7747Crossref PubMed Scopus (284) Google Scholar, 23Ruf W. Biochemistry. PubMed Scopus Google Scholar). to the human TF extracellular domain and to the phosphorylated or TF cytoplasmic domain were in (22Dorfleutner A. Ruf W. Blood. 2003; 102: 3998-4005Crossref PubMed Scopus (87) Google Scholar). PAR2 and PKCα and and were and PKCα and were from and were by L. Brass P.J. N. M. Brass L.F. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). nematode anticoagulant and P. Y. L. Y. Huang S. S. J. M. M. P.J. I. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, A. S. Ruf W. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) were by and kinase inactive PKCα were from Cell and hamster cells TF B.M. Ruf W. J. Clin. Invest. PubMed Google Scholar) were transiently by in of 1 with of PAR1 or PAR2 a in a gene were and of for and TF were described in (22Dorfleutner A. Ruf W. Blood. 2003; 102: 3998-4005Crossref PubMed Scopus (87) Google Scholar). Ser253 to Ala TF, by and PAR1 and PAR2 were was for and on umbilical vein endothelial cells were from or and in containing 1 human and were for and the were the cells were to for with the the were the of Xa, an of was in containing and the Xa concentrations were by of the on of and Cell cells with and TF was in the case of cells and cells or endothelial cells were directly in for with phosphorylation or to the domain (22Dorfleutner A. Ruf W. Blood. 2003; 102: 3998-4005Crossref PubMed Scopus (87) Google Scholar). surface expression of TF, or PAR2, the cells were with and in PAR the cells were with ternary TF-VIIa-Xa Xa, complex or PAR agonist peptides for at expression was by on for PAR1 or PAR2 by and of PKCα from to were with ternary complex or for 1 and and with 1 cells were by in 1 1 and Cell was by at for and the membrane and were by of the at M. J. Clin. Invest. 1997; PubMed Scopus Google Scholar). membrane were in the and the were by protein for with are of at are as the and TF Phosphorylation on PAR2 in signaling PKC, which in cells TF cytoplasmic domain phosphorylation (20Zioncheck T.F. Roy S. Vehar G.A. J. Biol. Chem. 1992; 267: 3561-3564Abstract Full Text PDF PubMed Google Scholar, 21Mody R.S. Carson S.D. Biochemistry. 1997; 36: 7869-7875Crossref PubMed Scopus (41) Google Scholar, 22Dorfleutner A. Ruf W. Blood. 2003; 102: 3998-4005Crossref PubMed Scopus (87) Google Scholar). In TF cells, the TF cytoplasmic domain was phosphorylated in response to demonstrating expression of relevant that However, PAR1 PAR2 induced TF cytoplasmic domain phosphorylation cells but not PAR2 (9Riewald M. Ruf W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7742-7747Crossref PubMed Scopus (284) Google Scholar). address the role of PAR2, cells were to human PAR2 (9Riewald M. Ruf W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7742-7747Crossref PubMed Scopus (284) Google Scholar) or PAR2 in which the was to PAR2 agonist induced TF cytoplasmic domain phosphorylation in cell TF-dependent signaling, we used as an to in the ternary TF-VIIa-Xa In the TF-VIIa-Xa VIIa is but Xa is and signaling which the of signaling in coagulation, Xa is on cells to VIIa and X (9Riewald M. Ruf W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7742-7747Crossref PubMed Scopus (284) Google Scholar). PAR2 agonist the TF-VIIa-Xa signaling complex induced TF cytoplasmic domain phosphorylation only in cells that expressed PAR2. Thus, cleavage of PAR2 by the ternary TF-VIIa-Xa complex induces TF cytoplasmic domain and forming the ternary complex is not to TF cytoplasmic domain phosphorylation in cells. the that the of phosphorylation in response to PAR1 agonist stimulation in cells was to expression of hamster cells were transiently with human PAR1 or human PAR2. expression was by of PAR1 or PAR2 in PAR agonist or ternary phosphorylation as as in of PAR1 or PAR2 are with of PAR1 or PAR2 activation by ternary complex signaling. However, only PAR2 expression in ternary TF cytoplasmic domain phosphorylation Thus, activating PAR1 as as PAR2, ternary complex signaling TF cytoplasmic domain phosphorylation specifically through PAR2 PAR2 to TF PAR1 was in cells, it whether PAR1 is not to TF phosphorylation. address this from that also other PARs (9Riewald M. Ruf W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7742-7747Crossref PubMed Scopus (284) Google Scholar, 19Camerer E. Kataoka H. Kahn M. Lease K. Coughlin S.R. J. Biol. Chem. 2002; 277: 16081-16087Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) were TF cytoplasmic domain phosphorylation is by of the cytoplasmic in endothelial cells (22Dorfleutner A. Ruf W. Blood. 2003; 102: 3998-4005Crossref PubMed Scopus (87) Google Scholar), and of the of TF cytoplasmic domain phosphorylation. were with to human to TF with PAR2 or expression in cells were with PAR1 or PAR2 agonist and with thrombin, or ternary complex as relevant proteases that TF phosphorylation was in cells, but TF cytoplasmic domain phosphorylation was in cells with PAR2 demonstrate that PAR1 is not in the signaling pathway that phosphorylates the of TF in the that was by with cytoplasmic domain found in cells that of TF be phosphorylated (22Dorfleutner A. Ruf W. Blood. 2003; 102: 3998-4005Crossref PubMed Scopus (87) Google Scholar). of phosphorylation of the of TF in this and other indicates that and thus a protein is also important for phosphorylation of the TF cytoplasmic domain in response to PAR2 TF Phosphorylation of evidence that expressed TF can be phosphorylated downstream of PAR2 signaling, we cells. this cell expressed of PAR2 but of PAR1 and TF cells were by directly PAR1 or PAR2 agonist peptides to the TF cytoplasmic domain phosphorylation was in cell with PAR2 but not in cells with PAR1 agonist phosphorylation in response to PAR2 agonist is the of cell PAR2 to Thus, in cells that TF, and PAR2, TF cytoplasmic domain phosphorylation specifically downstream of PAR2 signaling. TF Phosphorylation for in of PAR1 and PAR2 signaling in TF cytoplasmic domain phosphorylation was in primary with to TF and of PAR1 and PAR2. PAR1 and PAR2 not were to expression of PAR1 or PAR2. PAR activation was by by and stimulation with the ternary complex induced of receptors PAR1 and PAR2, by TF-dependent signaling was somewhat with the cleavage by thrombin and with in the cell activation of PAR1 or PAR1 not TF phosphorylation In contrast, PAR2 with TF in TF cytoplasmic domain phosphorylation of stimulation with ternary PAR2 or of PAR2 with to TF at as TF in TF cytoplasmic domain phosphorylation of stimulation and PAR1 stimulation was also not to phosphorylation of this not in TF phosphorylation between and TF were by for in expression TF was phosphorylated of was by specificity of TF cytoplasmic domain phosphorylation in response to other coagulation proteases was in PAR2 and to cells were for 1 to the for but concentrations of factor and activated protein C as as thrombin not TF phosphorylation are important PAR1 has been to PAR2 P.J. N. M. Brass L.F. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Whereas the TF-VIIa-Xa ternary complex TF cytoplasmic domain phosphorylation with to that of a PAR2 signaling by VIIa TF phosphorylation in In contrast, stimulation with VIIa and which activation of coagulation, induced TF cytoplasmic domain phosphorylation. In Xa was by a Xa Xa that PAR2. of ternary complex signaling is further in the of with cells for only with VIIa and X Xa in this but to TF phosphorylation in with Xa at concentrations as as In the the cells were with VIIa and X in the of the potent Xa Xa TF cytoplasmic domain (9Riewald M. Ruf W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7742-7747Crossref PubMed Scopus (284) Google Scholar) that in the ternary TF-VIIa-Xa coagulation initiation complex signaling is induced by Xa and not evidence that phosphorylation of the TF cytoplasmic domain as a of the activation of a specific signaling pathway the initiation of PAR2 PKCα to TF the induced by phosphorylation of the cytoplasmic Ser258 that is the target for a Pro-directed kinase (20Zioncheck T.F. Roy S. Vehar G.A. J. Biol. Chem. 1992; 267: 3561-3564Abstract Full Text PDF PubMed Google Scholar, 21Mody R.S. Carson S.D. Biochemistry. 1997; 36: 7869-7875Crossref PubMed Scopus (41) Google Scholar, 22Dorfleutner A. Ruf W. Blood. 2003; 102: 3998-4005Crossref PubMed Scopus (87) Google Scholar). However, phosphorylation of Ser253 to be for PMA-induced Ser258 phosphorylation in (22Dorfleutner A. Ruf W. Blood. 2003; 102: 3998-4005Crossref PubMed Scopus (87) Google Scholar). of the phosphorylation site Ser253 or the TF phosphorylation induced by ternary complex signaling TF cytoplasmic domain phosphorylation was also by for and and as the but not the further demonstrate we PKCα or to PKCα TF cytoplasmic domain phosphorylation was in cells inactive as with or PKCα cells Thus, PKCα is important for TF cytoplasmic domain phosphorylation downstream of PAR2, by the phosphorylation site at PAR1 signaling activates PKCα in endothelial cells to permeability D. A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, M. D. R.D. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar), but PKCα activation is TF cytoplasmic domain phosphorylation induced by PAR2 agonist or ternary complex signaling with somewhat delayed 1 and stimulation of from the to the membrane is for kinase J. 2003; PubMed Scopus Google Scholar). the of TF cytoplasmic domain phosphorylation and PKCα we the membrane of PKCα at the and that the ternary complex PKCα from the to the membrane but was only and not of stimulation of PAR1 cells. In contrast, the cells PAR2 not only of PKCα to the membrane but also sustained recruitment ternary complex signaling. of that the prolonged of PKCα was stimulation TF cytoplasmic domain phosphorylation in the of PAR2. stimulation of or endothelial cells also in prolonged PKCα evidence that the of PKCα at the membrane is an important for TF cytoplasmic domain phosphorylation. is how PAR2 signaling from PAR1 activation to sustained PKCα G protein-coupled receptor activation induces by phospholipase C to for membrane However, is also by which is to prolonged membrane recruitment of PubMed Scopus Google Scholar). the role of and in PKCα activation downstream of PAR2, the of specific on TF cytoplasmic domain phosphorylation was Whereas the specific was the ternary or PAR2 TF cytoplasmic domain phosphorylation and not further evidence that the pathway downstream of PAR2 to we the of on TF phosphorylation by PMA, which directly activates and only PMA-induced TF demonstrating that pathways downstream of are not by of on PKCα was to PKCα In PAR2 cells, the initial and sustained PKCα membrane recruitment in response to TF ternary complex signaling. to no on PKCα in PAR2 cells on of PKCα at 1 and by and the by was and In contrast, TF-dependent PAR1 activation in PKCα that was dependent on Thus, sustained activation of PKCα and TF cytoplasmic domain phosphorylation are dependent on a pathway downstream of PAR2 signaling. In this we show that PAR2 signaling specifically targets the TF cytoplasmic domain by TF phosphorylation. demonstrate that the ternary TF-VIIa-Xa complex activates PAR1 or PAR2, on of PAR and but only PAR2 signaling leads to TF phosphorylation. TF cytoplasmic domain phosphorylation is dependent on PKCα the that phosphorylation of the Ser253 is for subsequent Ser258 which is by the to TF (22Dorfleutner A. Ruf W. Blood. 2003; 102: 3998-4005Crossref PubMed Scopus (87) Google Scholar). PKCα membrane recruitment downstream of PAR1 or PAR2 receptor activation by the ternary However, only PAR2 signaling induced sustained PKCα membrane which coincides with the somewhat delayed of TF phosphorylation. that PAR1 and PAR2 PKCα through distinct activation of PKCα downstream of PAR2 is dependent on which is to prolonged membrane of PubMed Scopus Google Scholar). that of PAR2, induces TF phosphorylation and sustained PKCα membrane recruitment further for an essential role of prolonged PKCα signaling in TF cytoplasmic domain phosphorylation. This study to evidence (10Riewald M. Petrovan R.J. Donner A. Mueller B.M. Ruf W. Science. 2002; 296: 1880-1882Crossref PubMed Scopus (755) Google Scholar, S.W. Chipiuk A. Nelson R.C. Hollenberg M.D. Murray A.G. Circ. Res. 2003; 92: 272-278Crossref PubMed Scopus (100) Google Scholar, 13Vouret-Craviari V. Grall D. Van Obberghen-Schilling E. J. Thromb. Haemostasis. 2003; 1: 1103-1111Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) that PAR1 and PAR2 signaling elicit specific and only partially responses in endothelial cells. More importantly, only TF-dependent signaling, but not the signaling by other coagulation induced cytoplasmic domain phosphorylation. phosphorylation of the TF cytoplasmic domain thus a for upstream coagulation signaling through PAR2. PAR2 and TF are induced by in endothelial cells in tissue C.T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, S. V. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and TF has been in vivo in endothelial cells in inflammation J. A. J. Google Scholar) or in endothelium J. PubMed Scopus Google Scholar). PAR2 is found to be of R. J.R. M. L. Blood. 2003; 102: PubMed Scopus Google Scholar), which also Thus, phosphorylation of TF roles in the biology and of vascular cell that TF and PAR2. role of the TF cytoplasmic domain has been in heterologous of the cytoplasmic domain has on the of TF B.M. Ruf W. J. Clin. Invest. PubMed Google Scholar, M. M. Res. 2000; PubMed Scopus Google Scholar, S.D. Thromb. Haemostasis. 2000; PubMed Scopus Google Scholar) and on TF-VIIa signaling, as by immediate early gene induction or phosphorylation events downstream of G protein-coupled receptor signaling E. E. K. H. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Rao L.V.M. M. Petersen L.C. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Van Petersen L.C. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). Although the relevant PARs that TF-dependent signaling in cell are studies are with an that the TF cytoplasmic domain is not for PAR show that TF ternary complex signaling in the case of PAR1 and PAR2 signaling is to PKCα membrane recruitment 1 However, TF cytoplasmic domain phosphorylation is a delayed and it is not that the TF cytoplasmic domain no role in early responses of TF-VIIa signaling. In addition, TF cytoplasmic domain phosphorylation to be by ternary TF-VIIa-Xa in with TF-VIIa at which that the TF cytoplasmic domain plays a role in signaling with activation of the coagulation TF is important for B.M. Ruf W. J. Clin. Invest. PubMed Google Scholar, A. A. Proc. Natl. Acad. Sci. U. S. A. 92: PubMed Scopus Google Scholar), cell and I. Y. Mueller B.M. Ruf W. J. Cell Biol. PubMed Scopus Google Scholar, Riewald M. Huang Y. Y. Mueller B.M. Ruf W. J. Clin. Invest. 1999; PubMed Scopus Google Scholar, S. V. Blood. 92: PubMed Google Scholar, A. M. C. M. L. Blood. 2000; PubMed Google Scholar). Whereas PAR1 signaling induces activation (12Klarenbach S.W. Chipiuk A. Nelson R.C. Hollenberg M.D. Murray A.G. Circ. Res. 2003; 92: 272-278Crossref PubMed Scopus (100) Google Scholar, 13Vouret-Craviari V. Grall D. Van Obberghen-Schilling E. J. Thromb. Haemostasis. 2003; 1: 1103-1111Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), PAR2 only transiently activates activating and a β-arrestin scaffolding complex for (14DeFea K.A. Zalevsky J. Thoma M.S. Déry O. Mullins R.D. Bunnett N. J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (696) Google Scholar, 15Ge L. Ly Y. Hollenberg M. DeFea K. J. Biol. Chem. 2003; 278: 34418-34426Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). pathways cell a that is possibly by PAR2 signaling. PAR2 signaling that targets the TF cytoplasmic domain in this and the the by which PAR2 signaling the receptor TF that PAR cleavage by Phosphorylation of the TF cytoplasmic domain the recruitment of and thus and nonhemostatic roles of TF in cell angiogenesis, and inflammation. and Donner for for the of A. R. L. and A. for and M. for