title: Plasma protein S residues 37-50 mediate its binding to factor Va and inhibition of blood coagulation.
abstract: Protein S (PS) is an anticoagulant plasma protein whose deficiency is associated with increased risk of venous thrombosis. PS directly inhibits thrombin generation by the blood coagulation pathways by several mechanisms, including by binding coagulation factors (F) Va and Xa. To identify PS sequences that mediate inhibition of FVa activity, antibodies and synthetic peptides based on PS sequence were prepared and employed in plasma coagulation assays, purified component prothrombinase assays, binding assays, and immunoblots. In the absence of activated protein C, monoclonal antibody (Mab) S4 shortened FXa-induced clotting in normal plasma but not in PS-depleted plasma. Mab S4 also blocked PS inhibition of FVa-dependent prothrombinase activity in purified component assays in the absence or presence of phospholipids and inhibited binding of PS to immobilised FVa. Epitope mapping identified N-terminal region residues 37-67 of PS as this antibody's epitope. A peptide representing PS residues 37-50 inhibited FVa-dependent prothrombinase activity in a non-competitive manner, with 50% inhibition observed at 11 µM peptide, whereas a peptide with a D-amino acid sequence of 37-50 was ineffective. FVa, but not FXa, bound specifically to the immobilised peptide representing residues 37-50, and the peptide inhibited binding of FVa to immobilised PS. These data implicate PS residues 37-50 as a binding site for FVa that mediates, at least in part, the direct inhibition of FVa-dependent procoagulant activity by PS.
authors.affiliation: Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037, USA. heeb@scripps.edu
authors.forename: Mary J
authors.initials: MJ
authors.lastname: Heeb
authors.forename: Rolf M
authors.initials: RM
authors.lastname: Mesters
authors.forename: José A
authors.initials: JA
authors.lastname: Fernández
authors.forename: Tilman M
authors.initials: TM
authors.lastname: Hackeng
authors.forename: Ryon K
authors.initials: RK
authors.lastname: Nakasone
authors.forename: John H
authors.initials: JH
authors.lastname: Griffin
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000925:Anticoagulants / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D001665:Binding Sites; D001777:Blood Coagulation; D018604:Epitope Mapping; D015943:Factor Va / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D017293:Protein S / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D013925:Thromboplastin / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000925:Anticoagulants; D010446:Peptide Fragments; D017293:Protein S; D015943:Factor Va; D013925:Thromboplastin; D015951:Factor Xa
references.citation: Blood. 2011 Jun 16;117(24):6685-93
references.pmid: 21508412
references.citation: J Biol Chem. 1980 Jan 10;255(1):274-83
references.pmid: 7350159
references.citation: Haematologica. 2009 Dec;94(12):1721-31
references.pmid: 19815836
references.citation: Pathophysiol Haemost Thromb. 2010;37(1):17-23
references.pmid: 20501981
references.citation: Thromb Haemost. 1999 Dec;82(6):1627-33
references.pmid: 10613646
references.citation: Thromb Haemost. 2007 Sep;98(3):543-56
references.pmid: 17849042
references.citation: J Thromb Haemost. 2010 Sep;8(9):1976-85
references.pmid: 20492471
references.citation: Blood. 2005 Jan 1;105(1):122-30
references.pmid: 15308562
references.citation: Blood. 1984 Dec;64(6):1297-300
references.pmid: 6238642
references.citation: FASEB J. 2009 Jul;23(7):2244-53
references.pmid: 19244162
references.citation: J Comput Aided Mol Des. 1997 May;11(3):293-304
references.pmid: 9263856
references.citation: Biochim Biophys Acta. 1995 Dec 12;1272(3):159-67
references.pmid: 8541347
references.citation: J Biol Chem. 1994 Aug 19;269(33):21051-8
references.pmid: 8063724
references.citation: J Biol Chem. 1993 Feb 5;268(4):2872-7
references.pmid: 8428962
references.citation: J Biol Chem. 1988 Oct 15;263(29):14884-90
references.pmid: 2971660
references.citation: Blood. 2009 Sep 10;114(11):2307-14
references.pmid: 19567881
references.citation: J Thromb Haemost. 2004 Oct;2(10):1766-73
references.pmid: 15456488
references.citation: J Biol Chem. 1998 Oct 16;273(42):27449-58
references.pmid: 9765274
references.citation: Blood. 1985 Dec;66(6):1452-5
references.pmid: 2933098
references.citation: FASEB J. 2013 Jul;27(7):2918-25
references.pmid: 23580615
references.citation: Thromb Haemost. 2013 Feb;109(2):229-37
references.pmid: 23238804
references.citation: J Biol Chem. 1999 Dec 17;274(51):36187-92
references.pmid: 10593904
references.citation: J Biol Chem. 2008 Nov 28;283(48):33046-52
references.pmid: 18784085
references.citation: J Mol Biol. 1999 Oct 29;293(3):653-65
references.pmid: 10543957
references.citation: Circulation. 2008 Oct 14;118(16):1659-67
references.pmid: 18824642
references.citation: Blood. 1991 Jun 15;77(12):2641-8
references.pmid: 2043766
references.citation: Protein Sci. 1996 Sep;5(9):1883-9
references.pmid: 8880912
references.citation: J Biol Chem. 1994 Jul 22;269(29):18735-8
references.pmid: 8034625
references.citation: J Clin Invest. 2009 Oct;119(10):2942-53
references.pmid: 19729839
references.citation: Blood Cells Mol Dis. 2002 Sep-Oct;29(2):190-9
references.pmid: 12490286
references.citation: J Thromb Haemost. 2006 Oct;4(10):2215-22
references.pmid: 16824189
references.citation: N Engl J Med. 1984 Dec 13;311(24):1525-8
references.pmid: 6239102
references.citation: Proc Natl Acad Sci U S A. 2006 Feb 28;103(9):3106-11
references.pmid: 16488980
references.citation: Thromb Haemost. 2012 Apr;107(4):690-8
references.pmid: 22370911
references.citation: Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2728-32
references.pmid: 8146182
references.citation: Int J Hematol. 2012 Apr;95(4):333-45
references.pmid: 22477541
references.citation: Blood. 2002 Oct 15;100(8):2812-9
references.pmid: 12351389
references.citation: Lancet. 1990 Jan 6;335(8680):61-2
references.pmid: 1967377
references.citation: Thromb Res. 2010 Jun;125(6):529-32
references.pmid: 20181378
references.citation: Semin Thromb Hemost. 1984 Apr;10(2):131-8
references.pmid: 6234659
references.citation: Int J Hematol. 2011 Aug;94(2):150-155
references.pmid: 21811774
references.citation: Thromb Haemost. 1997 Jun;77(6):1201-14
references.pmid: 9241758