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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
› Institutsangaben 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).
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Publikationsverlauf

Received: 29. September 2014

Accepted after major revision: 05. Januar 2015

Publikationsdatum:
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.


 

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