The Kunitz-3 domain of TFPI-α is required for protein S-dependent enhancement of factor Xa inhibition

Protein S (PS) enhances the inhibition of factor Xa (FXa) by tissue factor pathway inhibitor-alpha (TFPI-alpha) in the presence of Ca2+ and phospholipids. Altered forms of recombinant TFPI-alpha were used to determine the structures within TFPI-alpha that may be involved in this PS-dependent effect. Wild-type TFPI-alpha (TFPIWT), TFPI-alpha lacking the K3 domain (TFPI-(Delta K3)), and TFPI-alpha containing a single amino acid change at the putative P1 residue of K3 (R199L, TFPIK3P1) produced equivalent FXa inhibition in the absence of PS, whereas the response in FXa inhibition produced by PS was reduced with TFPIK3P1 (EC50 61.8 +/- 13.4nM vs 8.0 +/- 0.4nM for TFPIWT) and not detectable with TFPI-(Delta K3). Ligand blotting and surface plasmon resonance experiments demonstrated that FXa bound TFPIWT and TFPI-(Delta K3) but not the isolated K3 domain, whereas PS bound TFPIWT and the K3 domain but not TFPI-(Delta K3). Addition of TFPIWT, TFPIK3P1, or TFPI-(Delta K3) produced comparable prolongation of FXa-induced coagulation in PS-deficient plasma, but the anticoagulant effect of TFPIWT was substantially greater than that of TFPIK3P1 > TFPI-(Delta K3) in normal plasma and PS-deficient plasma reconstituted with PS. We conclude that the PS-mediated enhancement of FXa inhibition by TFPI-alpha involves an interaction between PS and TFPI-alpha, which requires the K3 domain of TFPI-alpha. (Blood. 2010; 116(8): 1344-1351)