The extrinsic pathway of coagulation is regulated by tissue factor pathway inhibitor (TFPI) through binding and inhibition of factor (F)Xa and tissue factor (TF)-FVIIa-FXa. Its cofactor, protein S increases the affinity for the TFPI-FXa binding. The TFPI-protein S interaction involves the laminin G-type 1 (LG1) subunit of the protein S sex hormone-binding globulin (SHBG)-like domain. The LG1 subunit also interacts with C4BP via its β-chain, which impairs the protein S cofactor functions. This could be explained by either overlapping binding sites or sterical hindrance by C4BP.
I aimed to determine functional binding sites for TFPI and C4BP within protein S LG1. I created protein S variants with additional N-linked glycans to locate the interaction site for both proteins. In plasma based thrombin generation assays, D253T and Q427N/K429T variants displayed decreased TFPI cofactor activity. Next, I generated alanine composite variants by substituting clusters of surface exposed residues at the location of the glycan attachments. The LG1B variant (K255A/E257A/D287A/R410A/K423A/E424A) exhibited severly reduced TFPI cofactor function in plasma and FXa inhibition assays while retaining normal activated protein C (APC) cofactor function and C4BP binding affinity.
Two N-linked glycan variants displayed modestly decreased C4BP binding affinity. The introduced glycans in L379T and G418N were located close to each other in LG1 and these regions cooperated in the C4BP binding.
I also investigated the TFPI cofactor functions of the protein S-C4BP and protein S-C4BP β-chain complexes. The protein S-C4BP exhibited reduced cofactor activity in CAT whilst protein S-β-chain enhanced TFPI in CAT and FXa inhibition assays normally.
In summary, I identified a potential TFPI interaction site, involving protein S residues Lys255, Glu257, Asp287, Arg410, Lys423 and Glu424. I also identified an important region for C4BP interaction in LG1. Additionally, my results suggest sterical hindrance by C4BP for decreased TFPI cofactor functions of protein S.