The thrombin-sensitive region of protein S mediates phospholipid-dependent interaction with factor Xa.
To test the hypothesis that factor Xa (fXa) interacts with protein S, fXa was labeled active-site specifically with a dansyl (D) dye via a Glu-Gly-Arg (EGR) tether to yield DEGR-fXa(i). When protein S was added to phosphatidylcholine/phosphatidylserine (PC/PS, 4:1) vesicle-bound DEGR-fXa(i), the anisotropy of the dansyl moiety was altered from 0.219 +/- 0.002 to 0.245 +/- 0.003. This change in dansyl anisotropy was not observed when DEGR-Xa(i) was titrated with protein S in the absence of PC/PS vesicles, or in the presence of 100% PC vesicles, or when PC/PS vesicle-bound DEGR-fXa(i) was titrated with thrombin-cleaved protein S. The protein S-dependent dansyl fluorescence change was specific for fXa because it was not observed for two homologous and similarly labeled DEGR-fIXa(i) and DEGR-fVIIa(i). Furthermore, protein S specifically and saturably altered the fluorescence anisotropy of PC/PS-bound active site-labeled LWB-FPR-fXa(i) (Kd = 33 nm) and was photocross-linked to PC/PS-bound LWB-FPR-fXa(i) analog, independently confirming the above results. Chemically synthesized microprotein S, comprising residues 1-116 of protein S and including the gamma-carboxyglutamic-rich domain, the thrombin-sensitive region (TSR), and the first epidermal growth factor-like domain (EGF1) of protein S, altered the anisotropy of PC/PS-bound DEGR-fXa(i) from 0.219 to 0.242, similar to the effect of the protein S titration (Kd = 303 nm), suggesting that microprotein S binds to DEGR-fXa(i). To identify individual protein S domain(s) that binds DEGR-fXa(i), the EGF1 and TSR domains were chemically synthesized and studied. The TSR altered the anisotropy of DEGR-fXa(i) by approximately 16% (Kd = 3.9 microm), but the EGF1 domain had no effect on the signal. In controls, the TSR domain did not alter the anisotropy of DEGR-fIXa(i) and DEGR-fVIIa(i), respectively. These data demonstrate that membrane-bound fXa binding to protein S involves the TSR of protein S.
Yegneswaran
Subramanian
S
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
Hackeng
Tilman M
TM
Dawson
Philip E
PE
Griffin
John H
JH
D015951:Factor Xa / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D008567:Membranes, Artificial; D010713:Phosphatidylcholines / Q000737:chemistry* / Q000378:metabolism; D010718:Phosphatidylserines / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017293:Protein S / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism
D005456:Fluorescent Dyes; D008567:Membranes, Artificial; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D017293:Protein S; D013917:Thrombin; D015951:Factor Xa
Thromb Haemost. 1996 Sep;76(3):397-403
8883277
Biophys J. 1997 Nov;73(5):2638-52
9370458
J Thromb Haemost. 2006 Feb;4(2):385-91
16420570
J Thromb Haemost. 2006 Oct;4(10):2215-22
16824189
J Biol Chem. 1987 Sep 25;262(27):12953-61
3477541
Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2728-32
8146182
Biochemistry. 2001 Jul 31;40(30):8852-60
11467946
Biochim Biophys Acta. 1995 Dec 12;1272(3):159-67
8541347
Chem Biol. 2002 Apr;9(4):485-94
11983337
J Biol Chem. 1994 Aug 19;269(33):21051-8
8063724
Thromb Haemost. 1999 Jul;82(1):80-7
10456458
J Biol Chem. 1991 Dec 5;266(34):23016-21
1660464
J Biol Chem. 1992 Aug 25;267(24):17012-21
1512240
Thromb Haemost. 1998 Dec;80(6):930-5
9869163
Biophys J. 2003 Feb;84(2 Pt 1):1238-51
12547804
J Biol Chem. 1997 Oct 3;272(40):25013-21
9312108
J Thromb Haemost. 2008 Jun;6(6):1044-6
18419747
Biophys J. 1996 Jun;70(6):2930-7
8744331
Biopolymers. 1998 Aug;46(2):53-63
9664841
Biochem J. 1973 Apr;131(4):719-28
4352912
J Biol Chem. 1998 May 8;273(19):11521-6
9565566
Biochem J. 2000 Aug 1;349 Pt 3:757-64
10903136
Biochem J. 1973 Apr;131(4):739-48
4352914
J Biol Chem. 1996 Nov 8;271(45):28168-75
8910432
J Mol Biol. 1999 Oct 29;293(3):653-65
10543957
Blood. 2005 Sep 1;106(5):1884; author reply 1884-5
16113232
Int J Pept Protein Res. 1992 Sep-Oct;40(3-4):180-93
1478777
Thromb Haemost. 2007 Jul;98(1):90-6
17597997
Biochemistry. 2000 May 9;39(18):5468-73
10820019
Proc Natl Acad Sci U S A. 2006 Feb 28;103(9):3106-11
16488980
Biochemistry. 1990 Feb 20;29(7):1757-63
2110000
Proc Natl Acad Sci U S A. 2000 Dec 19;97(26):14074-8
11106381