Key result
The protein S-mediated enhancement of factor Xa inhibition by TFPI-α requires the K3 domain, as deletion abolishes the effect and a point mutation reduces it (EC50 61.8 nM vs 8.0 nM for wild-type).
Absolute Event Rate: 61.8% vs 8%
The Kunitz-3 domain of TFPI-alpha is essential for the Protein S-dependent enhancement of factor Xa inhibition, highlighting a specific structural requirement for this anticoagulant mechanism.
K3 domain targeting should not yet guide therapy; leaves open in vivo thrombosis validation and human translation.
Protein S (PS) enhances the inhibition of factor Xa (FXa) by tissue factor pathway inhibitor-alpha (TFPI-alpha) in the presence of Ca(2+) 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 (TFPI(WT)), TFPI-alpha lacking the K3 domain (TFPI-(DeltaK3)), and TFPI-alpha containing a single amino acid change at the putative P1 residue of K3 (R199L, TFPI(K3P1)) produced equivalent FXa inhibition in the absence of PS, whereas the response in FXa inhibition produced by PS was reduced with TFPI(K3P1) (EC(50) 61.8 +/- 13.4nM vs 8.0 +/- 0.4nM for TFPI(WT)) and not detectable with TFPI-(DeltaK3). Ligand blotting and surface plasmon resonance experiments demonstrated that FXa bound TFPI(WT) and TFPI-(DeltaK3) but not the isolated K3 domain, whereas PS bound TFPI(WT) and the K3 domain but not TFPI-(DeltaK3). Addition of TFPI(WT), TFPI(K3P1), or TFPI-(DeltaK3) produced comparable prolongation of FXa-induced coagulation in PS-deficient plasma, but the anticoagulant effect of TFPI(WT) was substantially greater than that of TFPI(K3P1) > TFPI-(DeltaK3) 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.
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Ndonwi et al. (2010) studied this question. Altered forms of recombinant TFPI-α (TFPI-ΔK3, TFPIK3P1) vs. Wild-type TFPI-α (TFPIWT) was evaluated on Protein S-dependent enhancement of factor Xa inhibition (EC50 in nM). The protein S-mediated enhancement of factor Xa inhibition by TFPI-α requires the K3 domain, as deletion abolishes the effect and a point mutation reduces it (EC50 61.8 nM vs 8.0 nM for wild-type).
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