THROMBOSIS AND HEMOSTASIS The Kunitz-3 domain of TFPI- 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(TFPI) in the presence of Ca2 and phospholipids. Altered forms of recombinant TFPIwere used to determine the structures within TFPIthat may be involved in this PS-dependent effect. Wild-type TFPI(TFPIWT), TFPIlacking the K3 domain (TFPIK3), and TFPIcontaining 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 TFPIK3. Ligand blotting and surface plasmon resonance experiments demonstrated that FXa bound TFPIWT and TFPIK3 but not the isolated K3 domain, whereas PS bound TFPIWT and the K3 domain but not TFPIK3. Addition of TFPIWT, TFPIK3P1, or TFPIK3 produced comparable prolongation of FXa-induced coagulation in PS-deficient plasma, but the anticoagulant effect of TFPIWT was substantially greater than that of TFPIK3P1 > TFPIK3 in normal plasma and PS-deficient plasma reconstituted with PS. We conclude that the PS-mediated enhancement of FXa inhibition by TFPIinvolves an interaction between PS and TFPI, which requires the K3 domain of TFPI. (Blood. 2010;116(8):1344-1351)