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Thromb Haemost 2015; 113(05): 976-987
DOI: 10.1160/TH14-09-0803
DOI: 10.1160/TH14-09-0803
Coagulation and Fibrinolysis
Amino acid residues in the laminin G domains of protein S involved in tissue factor pathway inhibitor interaction
Financial support: This work was supported by the Swedish Research Council (grant 71430), grants from the Swedish Heart and Lung Foundation, Söderberg’s foundation, the Alfred Österlund’s Foundation, research funds from the University Hospital in Malmö and the British Heart Foundation (FS/12/60/29874).
Further Information
Publication History
Received:
29 September 2014
Accepted after major revision:
05 January 2015
Publication Date:
24 November 2017 (online)
Summary
Protein S functions as a cofactor for tissue factor pathway inhibitor (TFPI) and activated protein C (APC). The sex hormone binding globulin (SHBG)-like region of protein S, consisting of two laminin G-like domains (LG1 and LG2), contains the binding site for C4b-binding protein (C4BP) and TFPI. Furthermore, the LG-domains are essential for the TFPI-cofactor function and for expression of full APC-cofactor function. The aim of the current study was to localise functionally important interaction sites in the protein S LG-domains using amino acid substitutions. Four protein S variants were created in which clusters of surface-exposed amino acid residues within the LG-domains were substituted. All variants bound normally to C4BP and were fully functional as cofactors for APC in plasma and in pure component assays. Two variants, SHBG2 (E612A, I614A, F265A, V393A, H453A), involving residues from both LG-domains, and SHBG3 (K317A, I330A, V336A, D365A) where residues in LG1 were substituted, showed 50–60 % reduction in enhancement of TFPI in FXa inhibition assays. For SHBG3 the decreased TFPI cofactor function was confirmed in plasma based thrombin generation assays. Both SHBG variants bound to TFPI with decreased affinity in surface plasmon resonance experiments. The TFPI Kunitz 3 domain is known to contain the interaction site for protein S. Using in silico analysis and protein docking exercises, preliminary models of the protein S SHBG/TFPI Kunitz domain 3 complex were created. Based on a combination of experimental and in silico data we propose a binding site for TFPI on protein S, involving both LGdomains.
Keywords
Protein S - tissue factor pathway inhibitor - C4b binding protein - sex hormone binding globulin-like region - laminin G-like domain* Contributed equally to this work.
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References
- 1 Hackeng TM, Sere KM, Tans G. et al. Protein S stimulates inhibition of the tissue factor pathway by tissue factor pathway inhibitor. Proc Natl Acad Sci USA 2006; 103: 3106-3111.
- 2 Peraramelli S, Thomassen S, Heinzmann A. et al. Inhibition of tf: fviia-catalysed fix and fx activation by tfpi and tfpi constructs. J Thromb Haemost. 2014. Epub ahead of print.
- 3 Dahlback B, Stenflo J. High Molecular Weight Complex in Human Plasma between Vitamin K-Dependent Protein S and Complement Component C4b-Binding Protein. PNAS 1981; 78: 2512-2516.
- 4 Ahnstrom J, Andersson HM, Canis K. et al. Activated protein C cofactor function of protein S: a novel role for a gamma-carboxyglutamic acid residue. Blood 2011; 117: 6685-6693.
- 5 Andersson HM, Arantes MJ, Crawley JT. et al. Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain. Blood 2010; 115: 4878-4885.
- 6 Saller F, Villoutreix BO, Amelot A. et al. The gamma-carboxyglutamic acid domain of anticoagulant protein S is involved in activated protein C cofactor activity, independently of phospholipid binding. Blood. 2005. 105 01 122-130 Epub 2004/08/17.
- 7 Mille-Baker B, Rezende SM, Simmonds RE. et al. Deletion or replacement of the second EGF-like domain of protein S results in loss of APC cofactor activity. Blood 2003; 101: 1416-1418.
- 8 Saposnik B, Borgel D, Aiach M, Gandrille S. Functional properties of the sexhormone-binding globulin (SHBG)-like domain of the anticoagulant protein S. Eur J Biochem 2003; 270: 545-555.
- 9 Villoutreix BO, Dahlback B, Borgel D, Gandrille S, Muller YA. Three-dimensional model of the SHBG-like region of anticoagulant protein S: new structurefunction insights. Proteins 2001; 43: 203-216.
- 10 Giri TK, Linse S, Garcia de Frutos P. et al. Structural requirements of anticoagulant protein S for its binding to the complement regulator C4b-binding protein. J Biol Chem 2002; 277: 15099-15106.
- 11 Chang GT, Maas BH, Ploos van Amstel HK. et al. Studies of the interaction between human protein S and human C4b-binding protein using deletion variants of recombinant human protein S. Thromb Haemost 1994; 71: 461-467.
- 12 Fernandez JA, Griffin JH, Chang GT. et al. Involvement of amino acid residues 423–429 of human protein S in binding to C4b-binding protein. Blood Cells Mol Dis 1998; 24: 101-113.
- 13 Linse S, Hardig Y, Schultz DA. et al. A region of vitamin K-dependent protein S that binds to C4b binding protein (C4BP) identified using bacteriophage peptide display libraries. J Biol Chem 1997; 272: 14658-14665.
- 14 Nelson RM, Long GL. Binding of protein S to C4b-binding protein. Mutagenesis of protein S. J Biol Chem 1992; 267: 8140-8145.
- 15 Walker FJ. Characterisation of a synthetic peptide that inhibits the interaction between protein S and C4b-binding protein. J Biol Chem 1989; 264: 17645-17648.
- 16 Weinstein RE, Walker FJ. Enhancement of rabbit protein S anticoagulant cofactor activity in vivo by modulation of the protein S C4B binding protein interaction. J Clin Invest 1990; 86: 1928-1935.
- 17 Nyberg P, Dahlback B, Garcia de Frutos P. The SHBG-like region of protein S is crucial for factor V-dependent APC-cofactor function. FEBS Lett 1998; 433: 28-32.
- 18 Reglinska-Matveyev N, Andersson HM. et al. TFPI cofactor function of protein S: essential role of the protein S SHBG-like domain. Blood 2014; 123: 3979-3987.
- 19 Evenas P, Garcia de Frutos P, Nicolaes GA. et al. The second laminin G-type domain of protein S is indispensable for expression of full cofactor activity in activated protein C-catalysed inactivation of factor Va and factor VIIIa. Thromb Haemost 2000; 84: 271-277.
- 20 Carlsson S, Dahlback B. Dependence on vitamin K-dependent protein S for eukaryotic cell secretion of the beta-chain of C4b-binding protein. J Biol Chem 2010; 285: 32038-32046.
- 21 Hardig Y, Dahlback B. The amino-terminal module of the C4b-binding protein beta-chain contains the protein S-binding site. J Biol Chem 1996; 271: 20861-20867.
- 22 van de Poel RH, Meijers JC, Bouma BN. Interaction between protein S and complement C4b-binding protein (C4BP). Affinity studies using chimeras containing c4bp beta-chain short consensus repeats. J Biol Chem 1999; 274: 15144-15150.
- 23 van de Poel RH, Meijers JC, Dahlback B. et al. C4b-binding protein (C4BP) beta-chain Short Consensus Repeat-2 specifically contributes to the interaction of C4BP with protein S. Blood Cells Mol Dis 1999; 25: 279-286.
- 24 Evenas P, Garcia De Frutos P, Linse S. et al. Both G-type domains of protein S are required for the high-affinity interaction with C4b-binding protein. Eur J Biochem 1999; 266: 935-942.
- 25 Van Wijnen M, Stam JG, Chang GT. et al. Characterisation of mini-protein S, a recombinant variant of protein S that lacks the sex hormone binding globulinlike domain. Biochem J 1998; 330: 389-396.
- 26 Heeb MJ, Kojima Y, Rosing J. et al. C-terminal Residues 621–635 of Protein S Are Essential for Binding to Factor Va. pp. 36187-36192.
- 27 Ahnstrom J, Andersson HM, Hockey V. et al. Identification of functionally important residues in TFPI Kunitz domain 3 required for the enhancement of its activity by protein S. Blood 2012; 120: 5059-5062.
- 28 Ndonwi M, Tuley EA, Broze Jr. GJ. The Kunitz-3 domain of TFPI-alpha is required for protein S-dependent enhancement of factor Xa inhibition. Blood 2010; 116: 1344-1351.
- 29 Hillarp A, Dahlback B. Novel subunit in C4b-binding protein required for protein S binding. J Biol Chem 1988; 263: 12759-12764.
- 30 Sun YH, Shen L, Dahlback B. Gla domain-mutated human protein C exhibiting enhanced anticoagulant activity and increased phospholipid binding. Blood 2003; 101: 2277-2284.
- 31 Friedrich U, Blom AM, Dahlback B. et al. Structural and energetic characteristics of the heparin-binding site in antithrombotic protein C. J Biol Chem 2001; 276: 24122-24128.
- 32 Shen L, Shah AM, Dahlback B. et al. Enhancing the activity of protein C by mutagenesis to improve the membrane-binding site: studies related to proline-10. Biochemistry 1997; 36: 16025-16031.
- 33 Norstrom EA, Steen M, Tran S. et al. Importance of protein S and phospholipid for activated protein C-mediated cleavages in factor Va. J Biol Chem 2003; 278: 24904-24911.
- 34 Fujikawa K, Legaz ME, Davie EW. Bovine factors X 1 and X 2 (Stuart factor). Isolation and characterisation. Biochemistry 1972; 11: 4882-4891.
- 35 Nelsestuen GL, Suttie JW. The mode of action of vitamin K. Isolation of a peptide containing the vitamin K-dependent portion of prothrombin. Proc Natl Acad Sci USA 1973; 70: 3366-3370.
- 36 Lundblad RL, Uhteg LC, Vogel CN. et al. Preparation and partial characterisation of two forms of bovine thrombin. Biochem Biophys Res Commun 1975; 66 (02) 482-9.
- 37 Tran S, Norstrom E, Dahlback B. Effects of prothrombin on the individual activated protein C-mediated cleavages of coagulation factor Va. J Biol Chem 2008; 283: 6648-6655.
- 38 Villoutreix BO, Garcia de Frutos P, Lovenklev M. et al. SHBG region of the anticoagulant cofactor protein S: secondary structure prediction, circular dichroism spectroscopy, and analysis of naturally occurring mutations. Proteins 1997; 29: 478-491.
- 39 Huang B, Schroeder M. Using protein binding site prediction to improve protein docking. Gene 2008; 422: 14-21.
- 40 Mine S, Yamazaki T, Miyata T. et al. Structural mechanism for heparin-binding of the third Kunitz domain of human tissue factor pathway inhibitor. Biochemistry 2002; 41: 78-85.
- 41 Jimenez-Garcia B, Pons C, Fernandez-Recio J. pyDockWEB: a web server for rigid-body protein-protein docking using electrostatics and desolvation scoring. Bioinformatics 2013; 29: 1698-1699.
- 42 Dahlback B. Purification of human vitamin K-dependent protein S and its limited proteolysis by thrombin. Biochem J 1983; 209: 837-846.
- 43 Malm J, Cohen E, Dackowski W. et al. Expression of completely gamma-carboxylated and beta-hydroxylated recombinant human vitamin-K-dependent protein S with full biological activity. Eur J Biochem 1990; 187: 737-743.
- 44 Giri TK, Hillarp A, Hardig Y, Zoller B. et al. A new direct, fast and quantitative enzyme-linked ligandsorbent assay for measurement of free protein S antigen. Thromb Haemost 1998; 79: 767-772.
- 45 Dahlback B. Inhibition of protein Ca cofactor function of human and bovine protein S by C4b-binding protein. J Biol Chem 1986; 261: 12022-12027.
- 46 Norstrom E, Thorelli E, Dahlback B. Functional characterisation of recombinant FV Hong Kong and FV Cambridge. Blood 2002; 100: 524-530.
- 47 Dahlback B. Human coagluation factor V purification and thrombin-catalysed activation. J Clin Invest 1980; 66: 583-591.
- 48 Castoldi E, Simioni P, Tormene D. et al. Hereditary and acquired protein S deficiencies are associated with low TFPI levels in plasma. J Thromb Haemost 2010; 08: 294-300.
- 49 Wood JP, Ellery PE, Maroney SA. et al. Protein S is a cofactor for platelet and endothelial tissue factor pathway inhibitor-alpha but not for cell surface-associated tissue factor pathway inhibitor. Arterioscler Thromb Vasc Biol 2014; 34: 169-176.