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• W2077777862 abstract "The complex of factor VIIa (FVIIa) with tissue factor (TF) triggers coagulation by recognizing its macromolecular substrate factors IX (FIX) and X (FX) predominantly through extended exosite interactions. In addition, TF mediates unique cell-signaling properties in cancer, angiogenesis, and inflammation that involve proteolytic cleavage of protease-activated receptor 2 (PAR2). PAR2 is cleaved by FVIIa in the binary TF·FVIIa complex and by FXa in the ternary TF·FVIIa·FXa complex, but physiological roles of these signaling complexes are incompletely understood. In a screen of FVIIa protease domain mutants, three variants (Q40A, Q143N, and T151S) activated macromolecular coagulation substrates and supported signaling of the ternary TF·FVIIa-Xa complex normally but were severely impaired in binary TF·FVIIa·PAR2 signaling. The residues identified were located in the model-predicted S2′ pocket of FVIIa, and complementary PAR2 P2′ Leu-38 replacements demonstrated that the P2′ side chain was indeed crucial for PAR2 cleavage by TF·FVIIa. In addition, PAR2 was activated more efficiently by FVIIa T99Y, consistent with further contributions from the S2 subsite. The P2 residue preference of FVIIa and FXa predicted additional PAR2 mutants that were efficiently activated by TF·FVIIa but resistant to cleavage by the alternative PAR2 activator FXa. Thus, contrary to the paradigm of exosite-assisted cleavage of PAR1 by thrombin, the cofactor-associated protease FVIIa recognizes PAR2 predominantly by catalytic cleft interactions. Furthermore, the delineated molecular details of this substrate interaction enabled protein engineering of protease-selective PAR2 receptors that will aid further studies to dissect the roles of TF signaling complexes in vivo. The complex of factor VIIa (FVIIa) with tissue factor (TF) triggers coagulation by recognizing its macromolecular substrate factors IX (FIX) and X (FX) predominantly through extended exosite interactions. In addition, TF mediates unique cell-signaling properties in cancer, angiogenesis, and inflammation that involve proteolytic cleavage of protease-activated receptor 2 (PAR2). PAR2 is cleaved by FVIIa in the binary TF·FVIIa complex and by FXa in the ternary TF·FVIIa·FXa complex, but physiological roles of these signaling complexes are incompletely understood. In a screen of FVIIa protease domain mutants, three variants (Q40A, Q143N, and T151S) activated macromolecular coagulation substrates and supported signaling of the ternary TF·FVIIa-Xa complex normally but were severely impaired in binary TF·FVIIa·PAR2 signaling. The residues identified were located in the model-predicted S2′ pocket of FVIIa, and complementary PAR2 P2′ Leu-38 replacements demonstrated that the P2′ side chain was indeed crucial for PAR2 cleavage by TF·FVIIa. In addition, PAR2 was activated more efficiently by FVIIa T99Y, consistent with further contributions from the S2 subsite. The P2 residue preference of FVIIa and FXa predicted additional PAR2 mutants that were efficiently activated by TF·FVIIa but resistant to cleavage by the alternative PAR2 activator FXa. Thus, contrary to the paradigm of exosite-assisted cleavage of PAR1 by thrombin, the cofactor-associated protease FVIIa recognizes PAR2 predominantly by catalytic cleft interactions. Furthermore, the delineated molecular details of this substrate interaction enabled protein engineering of protease-selective PAR2 receptors that will aid further studies to dissect the roles of TF signaling complexes in vivo. IntroductionCoagulation factor VIIa (FVIIa) 2The abbreviations used are: FVIIafactor VIIaFIXfactor IXFXfactor XTFtissue factorPARprotease-activated receptorERKextracellular-regulated kinaseTR3TR3 orphan receptorNAPc2nematode anticoagulant protein C2wtwild typeHaCaThuman immortalized keratinocyteHUVECumbilical vein endothelial cellDMEMDulbecco's modified Eagle's mediumPBSphosphate-buffered salineCHOChinese hamster ovaryIL-8interlurkin-8. in complex with its cellular receptor tissue factor (TF) mediates activation of two pathways of major physiological importance: (i) initiation of blood coagulation by activation of coagulation factors IX and X (FIX and FX), and (ii) induction of cell signaling through protease-activated receptors (PARs). TF·FVIIa-dependent signaling primarily activates PAR2, which belongs to a family of four G-protein-coupled receptors activated by specific proteolytic cleavage of their N-terminal extracellular domain (1Camerer E. Huang W. Coughlin S.R. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 5255-5260Crossref PubMed Scopus (601) Google Scholar). Cleavage unmasks a new N terminus, which serves as a tethered ligand that induces transmembrane signaling (2Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2101) Google Scholar). Activation of PAR2 by the TF·FVIIa binary complex involves cellular pools of TF with low affinity for FVIIa, whereas high affinity cell surface TF mediates coagulation activation and the associated cell signaling of the ternary complex of TF·FVIIa·FXa (3Ahamed J. Versteeg H.H. Kerver M. Chen V.M. Mueller B.M. Hogg P.J. Ruf W. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 13932-13937Crossref PubMed Scopus (288) Google Scholar). In the latter complex, FXa is the primary activator for PAR2 (4Riewald M. Ruf W. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 7742-7747Crossref PubMed Scopus (281) Google Scholar). PAR2 triggers typical G-protein-coupled as well as β-arrestin-dependent signaling and thereby induces cytokine, chemokine, and growth factor expression and regulates protein synthesis, cell motility, proliferation, and apoptosis (5Schaffner F. Ruf W. Semin. Thromb. Hemost. 2008; 34: 147-153Crossref PubMed Scopus (56) Google Scholar).Although the TF pathway also induces thrombin-dependent PAR1 signaling and transcriptional responses are often similar following PAR1 and PAR2 activation (6Albrektsen T. Sørensen B.B. Hjortø G.M. Fleckner J. Rao L.V.M. Petersen L.C. J. Thromb. Haemost. 2007; 5: 1588-1597Crossref PubMed Scopus (88) Google Scholar), PAR2 supports biological responses that are distinct from PAR1. For example, TF-dependent angiogenesis in TF cytoplasmic domain-deleted mice requires PAR2, but not PAR1 (7Belting M. Dorrell M.I. Sandgren S. Aguilar E. Ahamed J. Dorfleutner A. Carmeliet P. Mueller B.M. Friedlander M. Ruf W. Nat. Med. 2004; 10: 502-509Crossref PubMed Scopus (309) Google Scholar, 8Uusitalo-Jarvinen H. Kurokawa T. Mueller B.M. Andrade-Gordon P. Friedlander M. Ruf W. Arterioscler. Thromb. Vasc. Biol. 2007; 27: 1456-1462Crossref PubMed Scopus (96) Google Scholar), and breast cancer development was delayed in mice that lack PAR2, but not PAR1 (9Versteeg H.H. Schaffner F. Kerver M. Ellies L.G. Andrade-Gordon P. Mueller B.M. Ruf W. Cancer Res. 2008; 68: 7219-7227Crossref PubMed Scopus (113) Google Scholar). TF·FVIIa binary complex activation of PAR2 can be specifically blocked with an antibody that minimally affects coagulation, and this antibody, but not a coagulation-blocking anti-TF antibody, prevented breast cancer growth and PAR2-dependent pregnancy complication in vivo (9Versteeg H.H. Schaffner F. Kerver M. Ellies L.G. Andrade-Gordon P. Mueller B.M. Ruf W. Cancer Res. 2008; 68: 7219-7227Crossref PubMed Scopus (113) Google Scholar, 10Versteeg H.H. Schaffner F. Kerver M. Petersen H.H. Ahamed J. Felding-Habermann B. Takada Y. Mueller B.M. Ruf W. Blood. 2008; 111: 190-199Crossref PubMed Scopus (260) Google Scholar, 11Redecha P. Franzke C.W. Ruf W. Mackman N. Girardi G. J. Clin. Invest. 2008; 118: 3453-3461PubMed Google Scholar). The ternary TF·FVIIa·FXa complex or FXa also activate PAR2, so do proteases other than coagulation proteases (12Ossovskaya V.S. Bunnett N.W. Physiol. Rev. 2004; 84: 579-621Crossref PubMed Scopus (914) Google Scholar, 13Ruf W. Mueller B.M. Semin. Thromb. Hemost. 2006; 32: 61-68Crossref PubMed Scopus (143) Google Scholar). The role of these signaling pathways in vivo remains unclear, and improved tools are required to define the specific roles of coagulation protease signaling via PAR2.Exosite-driven macromolecular substrate recognition is a common mechanism by which proteases of the coagulation system achieve their remarkable specificity (14Krishnaswamy S. J. Thromb. Haemost. 2005; 3: 54-67Crossref PubMed Scopus (127) Google Scholar). This also applies to the activation of macromolecular substrates by TF·FVIIa (15Baugh R.J. Dickinson C.D. Ruf W. Krishnaswamy S. J. Biol. Chem. 2000; 275: 28826-28833Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 16Dickinson C.D. Kelly C.R. Ruf W. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 14379-14384Crossref PubMed Scopus (179) Google Scholar, 17Shobe J. Dickinson C.D. Edgington T.S. Ruf W. J. Biol. Chem. 1999; 274: 24171-24175Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) where the initial encounter with FX is governed primarily by interactions involving exosites in FVIIa and TF (18Dickinson C.D. Kelly C.R. Ruf W. Circulation. 1996; 94: 1272Google Scholar, 19Norledge B.V. Petrovan R.J. Ruf W. Olson A.J. Proteins. 2003; 53: 640-648Crossref PubMed Scopus (55) Google Scholar). Subsequent engagement of the FVIIa active site with residues of FX flanking the scissile bond then leads to proteolytic conversion of FX to FXa. Typically, cleavage of macromolecular substrates only requires a proper fit of the primary specificity residues with complementary sites in the catalytic cleft, and these interactions make only minor contributions to macromolecular substrate affinity. Exosite engagement is also a prominent feature in PAR recognition. Cleavage of PAR1 and PAR3 by thrombin is facilitated by charge interactions between exosite I in thrombin and a hirudin-like acidic element located C-terminally in the tethered PAR sequence (20Liu L.W. Vu T.K. Esmon C.T. Coughlin S.R. J. Biol. Chem. 1991; 266: 16977-16980Abstract Full Text PDF PubMed Google Scholar). Disruption of this exosite by mutagenesis severely impairs thrombin binding and the rate of PAR1 activation (21Ayala Y.M. Cantwell A.M. Rose T. Bush L.A. Arosio D. Di Cera E. Proteins. 2001; 45: 107-116Crossref PubMed Scopus (101) Google Scholar, 22Vu T.K. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2650) Google Scholar). Although PAR4 does not carry a recognition sequence for thrombin's exosite I, extended interactions between thrombin and the coreceptor PAR3 support efficient thrombin-PAR4 signaling (23Bah A. Chen Z. Bush-Pec L.A. Mathews F.S. Di Cera E. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 11603-11608Crossref PubMed Scopus (61) Google Scholar, 24Weiss E.J. Hamilton J.R. Lease K.E. Coughlin S.R. Blood. 2002; 100: 3240-3244Crossref PubMed Scopus (162) Google Scholar). Unlike these PARs, PAR2 lacks distinct exosite recognition sequences, a feature that may be required for its role as a broadly responsive sensor for trypsin-like serine proteases.Here, we address the role of PAR2 in coagulation protease signaling and define the structural requirements for PAR2 activation by FVIIa. Mutational mapping of FVIIa demonstrates that the S2′ pocket plays a critical role in PAR2 cleavage. Point mutations in this region abolish receptor activation while maintaining essentially normal FIX and FX cleavage, indicating distinctly different modes of recognition of PAR2 and coagulation substrates by TF·FVIIa. Mutations of PAR2 confirm that efficiency of cleavage is primarily determined by complementary interactions of the catalytic cleft and that single point mutations are sufficient to generate PAR2 receptors that loose responsiveness to specific coagulation proteases. These data support novel approaches to further define the role of the promiscuous PAR2 receptor in coagulation signaling pathways.DISCUSSIONHere, we provide a mutational mapping of the protease domain of FVIIa to identify structural determinants for recognition and cleavage of its cognate substrates, FIX, FX, and PAR2. TF·FVIIa-catalyzed FIX and FX activation was largely unaffected by mutations in the catalytic cleft of FVIIa, consistent with the previously established concept that binding of the macromolecular substrate is highly dependent on exosite interactions that facilitate primary substrate recognition at the active site (15Baugh R.J. Dickinson C.D. Ruf W. Krishnaswamy S. J. Biol. Chem. 2000; 275: 28826-28833Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 17Shobe J. Dickinson C.D. Edgington T.S. Ruf W. J. Biol. Chem. 1999; 274: 24171-24175Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). In contrast, cleavage of PAR2 was highly sensitive to substitution of FVIIa residues in the active site cleft and its perimeter. Results with three independent signaling readouts for TF·FVIIa concordantly identified residues Gln-40, Asp-72, Gln-143, and Thr-151 as key determinants for PAR2 recognition, whereas Ala-39 and Ile-60b were found to play minor but significant roles. An unaffected FIX or FX activation is indicative of an intact structural arrangement of the active site, and the mutational effect on PAR2 activation can be interpreted as a specific effect on substrate recognition. Remarkably, the Q143N FVIIa variant did not activate PAR2, but had essentially normal pro-coagulant activity, demonstrating that these two activities can be completely dissected by protein engineering.PAR2 activation with FVIIa and PAR2 variants agrees well with the predictions made by the model in Fig. 1A, which describes the docking of the P5-P5′ fragment of PAR2 into the active cleft of FVIIa. The model identifies FVIIa residues Gln-40, Gln-143, and Thr-151 as essential elements of the S2′ pocket, which accommodates the side chain of PAR2 Leu-38 (P2′). Consistent with the model, recognition of PAR2 was shown to be sensitive both to modifications of FVIIa at the S2′ subsite and to complementary mutations of PAR2 at the P2′ subsite. PAR2 signaling was diminished by FVIIa mutations that eliminated direct hydrophobic contacts and even by subtle changes, such as the T151S mutation. We also showed a strict requirement for large bulky side chains Leu, Ile, or Phe in P2′ of PAR2 and that cleavage-resistant receptors were produced by mutations of P2′ Leu-38 to Ser, Thr, or Val. In addition, PAR2 activation was abolished by the D72N substitution in the 70–80 Ca2+-binding loop. Recent characterization of this variant and its inhibition by AT showed that it exhibited normal catalytic activity and inhibitor interaction. Furthermore, the crystal structure of the D72N FVIIa-AT complex showed that Asn-72 adopted a conformation very similar to that of Asp-72 in wt FVIIa (40Bjelke J.R. Olsen O.H. Fodje M. Svensson L.A. Bang S. Bolt G. Kragelund B.B. Persson E. J. Biol. Chem. 2008; 283: 25863-25870Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). No significant perturbation of the Ca2+-binding loop or the catalytic cleft region was observed, which could suggest that Asp-72 interacts with P5′ Lys-41 in PAR2 as predicted by the model.Additional evidence for critical contributions to PAR2 recognition comes from the Q217E and T99Y FVIIa variants that exhibited >2-fold enhanced PAR2 binary complex signaling. Gln-217 is located adjacent to the S3/4 binding pocket, and Q217E replacement may stabilize the interaction with PAR2 P3 Lys-34. Thr-99 is located at the entrance to the S2 pocket, and substrate profiling (29Larsen K.S. Østergaard H. Bjelke J.R. Olsen O.H. Rasmussen H.B. Christensen L. Kragelund B.B. Stennicke H.R. Biochem. J. 2007; 405: 429-438Crossref PubMed Scopus (23) Google Scholar, 41Backes B.J. Harris J.L. Leonetti F. Craik C.S. Ellman J.A. Nat. Biotechnol. 2000; 18: 187-193Crossref PubMed Scopus (232) Google Scholar, 42Harris J.L. Backes B.J. Leonetti F. Mahrus S. Ellman J.A. Craik C.S. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 7754-7759Crossref PubMed Scopus (469) Google Scholar) indicated that the small Thr-99 residue in wt FVIIa allows access of large side chains to the open S2 pocket and thereby creates a preference for β-branched and hydrophobic amino acids (Thr, Leu, and Phe). The bulkier Tyr-99 in FVIIa T99Y likely restricts such access and shifts the P2 specificity toward Gly (29Larsen K.S. Østergaard H. Bjelke J.R. Olsen O.H. Rasmussen H.B. Christensen L. Kragelund B.B. Stennicke H.R. Biochem. J. 2007; 405: 429-438Crossref PubMed Scopus (23) Google Scholar) explaining the increased rate of PAR2 cleavage and enhanced wt PAR2 signaling.The delineated critical complementary contacts of FVIIa and PAR2 allowed us to engineer the PAR2 cleavage by FXa and still maintain efficient activation by TF·FVIIa. Two such PAR2 mutants (P2 G35T and G35I) were remarkably resistant to activation by free and ternary-complexed FXa. Contrary to the exosite interaction-driven FIX and FX activation, TF·FVIIa binary complex signaling is highly dependent on direct interactions on the cell surface between the extracellular domain of PAR2 and the catalytic cleft of FVIIa. This also contrasts with the activation of PAR1 by thrombin, which is greatly facilitated by thrombin's exosite 1 interaction with a hirudin-like C-terminal element of PAR1 involving charge complementarities (21Ayala Y.M. Cantwell A.M. Rose T. Bush L.A. Arosio D. Di Cera E. Proteins. 2001; 45: 107-116Crossref PubMed Scopus (101) Google Scholar, 22Vu T.K. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2650) Google Scholar, 43Myles T. Le Bonniec B.F. Stone S.R. Eur. J. Biochem. 2001; 268: 70-77Crossref PubMed Scopus (39) Google Scholar). Disruption of this exosite interaction by mutagenesis in contrast to mutations in the primed subsite region of the active site (e.g. Arg-35 and Glu-39) severely reduced the rate of PAR1 activation (21Ayala Y.M. Cantwell A.M. Rose T. Bush L.A. Arosio D. Di Cera E. Proteins. 2001; 45: 107-116Crossref PubMed Scopus (101) Google Scholar, 22Vu T.K. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2650) Google Scholar, 43Myles T. Le Bonniec B.F. Stone S.R. Eur. J. Biochem. 2001; 268: 70-77Crossref PubMed Scopus (39) Google Scholar). PAR1 or PAR2 recognition by thrombin or TF·FVIIa thus depends on different mechanisms and structural regions of the respective proteases.In conclusion, the results presented herein provide new insight into the structural requirements for PAR2 activation by TF·FVIIa and offer clues to how specific protein-protein interactions contribute to TF·FVIIa-mediated PAR2 signaling that triggers a diversity of cellular responses in cancer, inflammation, and angiogenesis. By exploiting the predominantly active-site-driven interaction between PAR2 and its activating proteases, we demonstrate the feasibility of rationally optimizing the receptor recognition sequence to selectively reduce PAR2 activation by FXa to a minimum. The physiological relevance of TF·FVIIa·FXa or other FXa-mediated PAR2 signaling is currently incompletely understood. The FVIIa and PAR2 variants generated in this study provide important new tools to dissect the role of FVIIa-mediated PAR2 activation and to further characterize the relative importance of TF coagulation complex signaling pathways in vivo. IntroductionCoagulation factor VIIa (FVIIa) 2The abbreviations used are: FVIIafactor VIIaFIXfactor IXFXfactor XTFtissue factorPARprotease-activated receptorERKextracellular-regulated kinaseTR3TR3 orphan receptorNAPc2nematode anticoagulant protein C2wtwild typeHaCaThuman immortalized keratinocyteHUVECumbilical vein endothelial cellDMEMDulbecco's modified Eagle's mediumPBSphosphate-buffered salineCHOChinese hamster ovaryIL-8interlurkin-8. in complex with its cellular receptor tissue factor (TF) mediates activation of two pathways of major physiological importance: (i) initiation of blood coagulation by activation of coagulation factors IX and X (FIX and FX), and (ii) induction of cell signaling through protease-activated receptors (PARs). TF·FVIIa-dependent signaling primarily activates PAR2, which belongs to a family of four G-protein-coupled receptors activated by specific proteolytic cleavage of their N-terminal extracellular domain (1Camerer E. Huang W. Coughlin S.R. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 5255-5260Crossref PubMed Scopus (601) Google Scholar). Cleavage unmasks a new N terminus, which serves as a tethered ligand that induces transmembrane signaling (2Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2101) Google Scholar). Activation of PAR2 by the TF·FVIIa binary complex involves cellular pools of TF with low affinity for FVIIa, whereas high affinity cell surface TF mediates coagulation activation and the associated cell signaling of the ternary complex of TF·FVIIa·FXa (3Ahamed J. Versteeg H.H. Kerver M. Chen V.M. Mueller B.M. Hogg P.J. Ruf W. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 13932-13937Crossref PubMed Scopus (288) Google Scholar). In the latter complex, FXa is the primary activator for PAR2 (4Riewald M. Ruf W. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 7742-7747Crossref PubMed Scopus (281) Google Scholar). PAR2 triggers typical G-protein-coupled as well as β-arrestin-dependent signaling and thereby induces cytokine, chemokine, and growth factor expression and regulates protein synthesis, cell motility, proliferation, and apoptosis (5Schaffner F. Ruf W. Semin. Thromb. Hemost. 2008; 34: 147-153Crossref PubMed Scopus (56) Google Scholar).Although the TF pathway also induces thrombin-dependent PAR1 signaling and transcriptional responses are often similar following PAR1 and PAR2 activation (6Albrektsen T. Sørensen B.B. Hjortø G.M. Fleckner J. Rao L.V.M. Petersen L.C. J. Thromb. Haemost. 2007; 5: 1588-1597Crossref PubMed Scopus (88) Google Scholar), PAR2 supports biological responses that are distinct from PAR1. For example, TF-dependent angiogenesis in TF cytoplasmic domain-deleted mice requires PAR2, but not PAR1 (7Belting M. Dorrell M.I. Sandgren S. Aguilar E. Ahamed J. Dorfleutner A. Carmeliet P. Mueller B.M. Friedlander M. Ruf W. Nat. Med. 2004; 10: 502-509Crossref PubMed Scopus (309) Google Scholar, 8Uusitalo-Jarvinen H. Kurokawa T. Mueller B.M. Andrade-Gordon P. Friedlander M. Ruf W. Arterioscler. Thromb. Vasc. Biol. 2007; 27: 1456-1462Crossref PubMed Scopus (96) Google Scholar), and breast cancer development was delayed in mice that lack PAR2, but not PAR1 (9Versteeg H.H. Schaffner F. Kerver M. Ellies L.G. Andrade-Gordon P. Mueller B.M. Ruf W. Cancer Res. 2008; 68: 7219-7227Crossref PubMed Scopus (113) Google Scholar). TF·FVIIa binary complex activation of PAR2 can be specifically blocked with an antibody that minimally affects coagulation, and this antibody, but not a coagulation-blocking anti-TF antibody, prevented breast cancer growth and PAR2-dependent pregnancy complication in vivo (9Versteeg H.H. Schaffner F. Kerver M. Ellies L.G. Andrade-Gordon P. Mueller B.M. Ruf W. Cancer Res. 2008; 68: 7219-7227Crossref PubMed Scopus (113) Google Scholar, 10Versteeg H.H. Schaffner F. Kerver M. Petersen H.H. Ahamed J. Felding-Habermann B. Takada Y. Mueller B.M. Ruf W. Blood. 2008; 111: 190-199Crossref PubMed Scopus (260) Google Scholar, 11Redecha P. Franzke C.W. Ruf W. Mackman N. Girardi G. J. Clin. Invest. 2008; 118: 3453-3461PubMed Google Scholar). The ternary TF·FVIIa·FXa complex or FXa also activate PAR2, so do proteases other than coagulation proteases (12Ossovskaya V.S. Bunnett N.W. Physiol. Rev. 2004; 84: 579-621Crossref PubMed Scopus (914) Google Scholar, 13Ruf W. Mueller B.M. Semin. Thromb. Hemost. 2006; 32: 61-68Crossref PubMed Scopus (143) Google Scholar). The role of these signaling pathways in vivo remains unclear, and improved tools are required to define the specific roles of coagulation protease signaling via PAR2.Exosite-driven macromolecular substrate recognition is a common mechanism by which proteases of the coagulation system achieve their remarkable specificity (14Krishnaswamy S. J. Thromb. Haemost. 2005; 3: 54-67Crossref PubMed Scopus (127) Google Scholar). This also applies to the activation of macromolecular substrates by TF·FVIIa (15Baugh R.J. Dickinson C.D. Ruf W. Krishnaswamy S. J. Biol. Chem. 2000; 275: 28826-28833Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 16Dickinson C.D. Kelly C.R. Ruf W. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 14379-14384Crossref PubMed Scopus (179) Google Scholar, 17Shobe J. Dickinson C.D. Edgington T.S. Ruf W. J. Biol. Chem. 1999; 274: 24171-24175Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) where the initial encounter with FX is governed primarily by interactions involving exosites in FVIIa and TF (18Dickinson C.D. Kelly C.R. Ruf W. Circulation. 1996; 94: 1272Google Scholar, 19Norledge B.V. Petrovan R.J. Ruf W. Olson A.J. Proteins. 2003; 53: 640-648Crossref PubMed Scopus (55) Google Scholar). Subsequent engagement of the FVIIa active site with residues of FX flanking the scissile bond then leads to proteolytic conversion of FX to FXa. Typically, cleavage of macromolecular substrates only requires a proper fit of the primary specificity residues with complementary sites in the catalytic cleft, and these interactions make only minor contributions to macromolecular substrate affinity. Exosite engagement is also a prominent feature in PAR recognition. Cleavage of PAR1 and PAR3 by thrombin is facilitated by charge interactions between exosite I in thrombin and a hirudin-like acidic element located C-terminally in the tethered PAR sequence (20Liu L.W. Vu T.K. Esmon C.T. Coughlin S.R. J. Biol. Chem. 1991; 266: 16977-16980Abstract Full Text PDF PubMed Google Scholar). Disruption of this exosite by mutagenesis severely impairs thrombin binding and the rate of PAR1 activation (21Ayala Y.M. Cantwell A.M. Rose T. Bush L.A. Arosio D. Di Cera E. Proteins. 2001; 45: 107-116Crossref PubMed Scopus (101) Google Scholar, 22Vu T.K. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2650) Google Scholar). Although PAR4 does not carry a recognition sequence for thrombin's exosite I, extended interactions between thrombin and the coreceptor PAR3 support efficient thrombin-PAR4 signaling (23Bah A. Chen Z. Bush-Pec L.A. Mathews F.S. Di Cera E. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 11603-11608Crossref PubMed Scopus (61) Google Scholar, 24Weiss E.J. Hamilton J.R. Lease K.E. Coughlin S.R. Blood. 2002; 100: 3240-3244Crossref PubMed Scopus (162) Google Scholar). Unlike these PARs, PAR2 lacks distinct exosite recognition sequences, a feature that may be required for its role as a broadly responsive sensor for trypsin-like serine proteases.Here, we address the role of PAR2 in coagulation protease signaling and define the structural requirements for PAR2 activation by FVIIa. Mutational mapping of FVIIa demonstrates that the S2′ pocket plays a critical role in PAR2 cleavage. Point mutations in this region abolish receptor activation while maintaining essentially normal FIX and FX cleavage, indicating distinctly different modes of recognition of PAR2 and coagulation substrates by TF·FVIIa. Mutations of PAR2 confirm that efficiency of cleavage is primarily determined by complementary interactions of the catalytic cleft and that single point mutations are sufficient to generate PAR2 receptors that loose responsiveness to specific coagulation proteases. These data support novel approaches to further define the role of the promiscuous PAR2 receptor in coagulation signaling pathways." @default.
• W2077777862 created "2016-06-24" @default.
• W2077777862 creator A5005286957 @default.
• W2077777862 creator A5022194230 @default.
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• W2077777862 date "2010-06-01" @default.
• W2077777862 modified "2023-10-17" @default.
• W2077777862 title "Engineering of Substrate Selectivity for Tissue Factor·Factor VIIa Complex Signaling through Protease-activated Receptor 2" @default.
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