Engineering of substrate selectivity for tissue factor.factor VIIa complex signaling through protease-activated receptor 2.
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.
Larsen
Katrine S
KS
Department of Haemostasis Biochemistry, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark.
Ostergaard
Henrik
H
Olsen
Ole H
OH
Bjelke
Jais R
JR
Ruf
Wolfram
W
Petersen
Lars C
LC
D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D015942:Factor VIIa / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D046911:Macromolecular Substances / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D044464:Receptor, PAR-2 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013379:Substrate Specificity; D013925:Thromboplastin / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D046911:Macromolecular Substances; D044464:Receptor, PAR-2; D013925:Thromboplastin; D015942:Factor VIIa
Cell. 1991 Mar 22;64(6):1057-68
1672265
J Biol Chem. 1993 May 5;268(13):9780-6
7683662
Eur J Biochem. 2001 Jan;268(1):70-7
11121104
J Biol Chem. 1999 Aug 20;274(34):24171-5
10446191
Blood. 2004 Apr 15;103(8):3029-37
15070680
Proc Natl Acad Sci U S A. 2001 Jul 3;98(14):7742-7
11438726
Biochemistry. 1999 Aug 24;38(34):10940-8
10460149
Cancer Res. 2008 Sep 1;68(17):7219-27
18757438
Proc Natl Acad Sci U S A. 2007 Jul 10;104(28):11603-8
17606903
J Biol Chem. 2000 Sep 15;275(37):28826-33
10889208
Nat Biotechnol. 2000 Feb;18(2):187-93
10657126
Physiol Rev. 2004 Apr;84(2):579-621
15044683
Nat Med. 2004 May;10(5):502-9
15098027
Proteins. 2001 Nov 1;45(2):107-16
11562940
J Clin Invest. 2008 Oct;118(10):3453-61
18802482
Proc Natl Acad Sci U S A. 2006 Sep 19;103(38):13932-7
16959886
J Biol Chem. 2004 May 28;279(22):23038-44
15039423
J Biol Chem. 1991 Sep 15;266(26):16977-80
1654318
Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14379-84
8962059
J Biol Chem. 2008 Sep 19;283(38):25863-70
18640965
Biochem J. 2007 Aug 1;405(3):429-38
17456045
Proc Natl Acad Sci U S A. 2000 May 9;97(10):5255-60
10805786
Nature. 1996 Mar 7;380(6569):41-6
8598903
Am J Physiol Cell Physiol. 2003 May;284(5):C1319-29
12540381
Science. 2002 Jun 7;296(5574):1880-2
12052963
Proteins. 2003 Nov 15;53(3):640-8
14579355
J Thromb Haemost. 2005 Jan;3(1):54-67
15634266
Semin Thromb Hemost. 2008 Mar;34(2):147-53
18645919
J Biol Chem. 2002 Feb 1;277(5):3622-31
11723140
Blood. 2008 Jan 1;111(1):190-9
17901245
J Comput Chem. 2009 Jul 30;30(10):1545-614
19444816
Blood. 2002 Nov 1;100(9):3240-4
12384423
J Thromb Haemost. 2007 Aug;5(8):1588-97
17470200
Chem Rev. 2005 Mar;105(3):973-99
15755082
Nature. 2000 Sep 14;407(6801):258-64
11001069
Biochemistry. 1995 Jul 11;34(27):8701-7
7542028
J Biol Chem. 2000 May 5;275(18):13502-9
10788464
Arterioscler Thromb Vasc Biol. 2007 Jun;27(6):1456-62
17363687
Proc Natl Acad Sci U S A. 2000 Jul 5;97(14):7754-9
10869434
J Mol Biol. 1999 Feb 5;285(5):2089-104
9925787
Semin Thromb Hemost. 2006 Apr;32 Suppl 1:61-8
16673267
Eur J Biochem. 1992 Mar 1;204(2):433-51
1541261