title: Amino acid residues in the laminin G domains of protein S involved in tissue factor pathway inhibitor interaction.
abstract: 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 LG-domains.
authors.forename: Sofia
authors.initials: S
authors.lastname: Somajo
authors.forename: Josefin
authors.initials: J
authors.lastname: Ahnström
authors.forename: Juan
authors.initials: J
authors.lastname: Fernandez-Recio
authors.forename: Magdalena
authors.initials: M
authors.lastname: Gierula
authors.forename: Bruno O
authors.initials: BO
authors.lastname: Villoutreix
authors.affiliation: Björn Dahlbäck MD, PhD, Professor of Blood Coagulation Research, Lund University, Department of Translational Medicine, Division of Clinical Chemistry, Wallenberg laboratory, floor 6, University Hospital, Malmö, S-20502 Malmö, Sweden, E-mail: bjorn.dahlback@med.lu.se.
authors.forename: Björn
authors.initials: B
authors.lastname: Dahlbäck
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D015943:Factor Va / Q000737:chemistry; D006801:Humans; D007797:Laminin / Q000737:chemistry*; D008074:Lipoproteins / Q000037:antagonists & inhibitors*; D016296:Mutagenesis; D010314:Partial Thromboplastin Time; D011487:Protein Conformation; D017293:Protein S / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012738:Sex Hormone-Binding Globulin / Q000737:chemistry; D020349:Surface Plasmon Resonance; D013917:Thrombin / Q000737:chemistry
chemical_list: D000596:Amino Acids; C400567:LAMC2 protein, human; D007797:Laminin; D008074:Lipoproteins; D017293:Protein S; D011994:Recombinant Proteins; D012738:Sex Hormone-Binding Globulin; C089141:laminin gamma 1; C051928:lipoprotein-associated coagulation inhibitor; D015943:Factor Va; D013917:Thrombin
keywords: C4b binding protein; Protein S; laminin G-like domain; sex hormone binding globulin-like region; tissue factor pathway inhibitor