Key Points• The protein S SHBG-like domain and, more specifically, its LG1 subunit are important for binding and enhancement of TFPI.• TFPI binding to the protein S SHBG-like domain likely positions TFPI Kunitz domain 2 for optimal interaction with the active site of FXa.Protein S is a cofactor for tissue factor pathway inhibitor (TFPI), accelerating the inhibition of activated factor X (FXa). TFPI Kunitz domain 3 residue Glu226 is essential for enhancement of TFPI by protein S. To investigate the complementary functional interaction site on protein S, we screened 44 protein S point, composite or domain swap variants spanning the whole protein S molecule for their TFPI cofactor function using a thrombin generation assay. Of these variants, two protein S/growth arrestspecific 6 chimeras, with either the whole sex hormone-binding globulin (SHBG)-like domain (Val243-Ser635; chimera III) or the SHBG laminin G-type 1 subunit (Ser283-Val459; chimera I), respectively, substituted by the corresponding domain in growth arrest-specific 6, were unable to enhance TFPI. The importance of the protein S SHBG-like domain (and its laminin G-type 1 subunit) for binding and enhancement of TFPI was confirmed in FXa inhibition assays and using surface plasmon resonance. In addition, protein S bound to C4b binding protein showed greatly reduced enhancement of TFPI-mediated inhibition of FXa compared with free protein S. We show that binding of TFPI to the protein S SHBG-like domain enables TFPI to interact optimally with FXa on a phospholipid membrane. (Blood. 2014;123(25):3979-3987)
IntroductionTissue factor pathway inhibitor (TFPI) is a ;41 kDa Kunitz-type protease inhibitor that consists of an acidic amino-terminal polypeptide, followed by 3 tandem Kunitz-type domains (Kunitz domains 1, 2, and 3) and a basic carboxy (C)-terminal tail. 1 It circulates in plasma at a concentration of ;1.6 nM. 2,3 The majority (;80%) of plasma TFPI is truncated and bound to lipoproteins, ;5% is localized in storage granules within platelets, ;5% circulates as free truncated variants, and only around 10% is considered to be free full-length TFPI, 4 with this having the greatest anticoagulant activity. [5][6][7] TFPI and its cofactor protein S downregulate tissue factor (TF)-induced thrombin generation, and a deficiency of either protein has been linked to an increased risk of venous thrombosis. [8][9][10] TFPI specifically inhibits the initiation of coagulation through direct binding and inhibition of factor Xa (FXa) and, in a FXa-dependent manner, inhibition of the TF/factor VIIa (FVIIa) complex by forming a quarternary TF/FVIIa/FXa/TFPI complex. 11,12 The P1 residue in the Kunitz domain 2 of TFPI is required for binding to FXa, whereas the P1 residue in Kunitz domain 1 is required for binding and inhibition of TF/FVIIa. 13 The kinetic mechanism of FXa inhibition by TFPI is described as a 2-step process where TFPI first forms an immediate encounter complex with FXa (FXa/TFPI), followed by a slow isomerization into a final tight complex (FXa/TFPI*...