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Thromb Haemost 2015; 113(05): 976-987
DOI: 10.1160/TH14-09-0803
Coagulation and Fibrinolysis
Schattauer GmbH

Amino acid residues in the laminin G domains of protein S involved in tissue factor pathway inhibitor interaction

Sofia Somajo   *
1   Department of Translational Medicine, Division of Clinical Chemistry, Lund University, Skåne University Hospital, Malmö, Sweden
,
Josefin Ahnström   *
2   Centre for Haematology, Department of Medicine, Imperial College London, Hammersmith Hospital Campus, London, UK
,
Juan Fernandez-Recio
3   Protein Interactions and Docking Life Sciences Department, Barcelona Supercomputing Centre, Barcelona, Spain
,
Magdalena Gierula
2   Centre for Haematology, Department of Medicine, Imperial College London, Hammersmith Hospital Campus, London, UK
,
Bruno O. Villoutreix
4   Inserm Unit U973, Therapeutic molecules in silico (MTi), Lamarck Building, Paris, France
,
Björn Dahlbäck
1   Department of Translational Medicine, Division of Clinical Chemistry, Lund University, Skåne University Hospital, Malmö, Sweden
› Author Affiliations 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)

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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.


 

  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    Google Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    Google Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 14 Nelson RM, Long GL. Binding of protein S to C4b-binding protein. Mutagenesis of protein S. J Biol Chem 1992; 267: 8140-8145.
    PubMedGoogle Scholar
  • 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.
    PubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    PubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 29 Hillarp A, Dahlback B. Novel subunit in C4b-binding protein required for protein S binding. J Biol Chem 1988; 263: 12759-12764.
    PubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 34 Fujikawa K, Legaz ME, Davie EW. Bovine factors X 1 and X 2 (Stuart factor). Isolation and characterisation. Biochemistry 1972; 11: 4882-4891.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 39 Huang B, Schroeder M. Using protein binding site prediction to improve protein docking. Gene 2008; 422: 14-21.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 42 Dahlback B. Purification of human vitamin K-dependent protein S and its limited proteolysis by thrombin. Biochem J 1983; 209: 837-846.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 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.
    PubMedGoogle Scholar
  • 46 Norstrom E, Thorelli E, Dahlback B. Functional characterisation of recombinant FV Hong Kong and FV Cambridge. Blood 2002; 100: 524-530.
    CrossrefPubMedGoogle Scholar
  • 47 Dahlback B. Human coagluation factor V purification and thrombin-catalysed activation. J Clin Invest 1980; 66: 583-591.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar
  • 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.
    CrossrefPubMedGoogle Scholar