Somajo Sofia, Ahnström Josefin, Fernandez-Recio Juan, Gierula Magdalena, Villoutreix Bruno O, Dahlbäck Björn
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:
Thromb Haemost. 2015 May;113(5):976-87. doi: 10.1160/TH14-09-0803. Epub 2015 Feb 26.
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.
蛋白S作为组织因子途径抑制物（TFPI）和活化蛋白C（APC）的辅因子发挥作用。蛋白S的性激素结合球蛋白（SHBG）样区域由两个层粘连蛋白G样结构域（LG1和LG2）组成，包含C4b结合蛋白（C4BP）和TFPI的结合位点。此外，LG结构域对于TFPI辅因子功能以及完整APC辅因子功能的表达至关重要。本研究的目的是通过氨基酸替换来定位蛋白S LG结构域中功能重要的相互作用位点。创建了四种蛋白S变体，其中LG结构域内表面暴露的氨基酸残基簇被替换。所有变体均能正常结合C4BP，并且在血浆和纯组分测定中作为APC的辅因子具有完全功能。两种变体，即涉及两个LG结构域残基的SHBG2（E612A、I614A、F265A、V393A、H453A）和LG1中残基被替换的SHBG3（K317A、I330A、V336A、D365A），在FXa抑制试验中增强TFPI的能力降低了50 - 60%。对于SHBG3，基于血浆的凝血酶生成试验证实了其TFPI辅因子功能降低。在表面等离子体共振实验中，两种SHBG变体与TFPI的结合亲和力均降低。已知TFPI Kunitz 3结构域包含与蛋白S的相互作用位点。通过计算机分析和蛋白质对接实验，创建了蛋白S SHBG/TFPI Kunitz结构域3复合物的初步模型。基于实验数据和计算机数据的结合，我们提出了TFPI在蛋白S上的一个结合位点，涉及两个LG结构域。
