Tissue factor-independent inhibition of thrombin generation by tissue factor pathway inhibitor-[alpha]

Summary Background Tissue factor pathway inhibitor-[alpha] (TFPI[alpha]) inhibits factor Xa by forming a binary TFPI-FXa complex in a reaction that is stimulated by protein S. TF-FVIIa forms a quaternary complex with TFPI[alpha] and FXa, which shuts off the initiation of coagulation via the extrinsic pathway. Aim To investigate whether direct inhibition of FXa by TFPI[alpha] independently of TF plays a role in downregulating coagulation. Methods Inhibition of FXa by TFPI[alpha] in plasma was determined by measuring thrombin generation triggered with FXa, the FX activator from Russell's viper venom (RVV-X), FXIa, or FIXa. TF-independent anticoagulant activities of TFPI[alpha] and its cofactor, protein S, were quantified: (i) after neutralization of TFPI[alpha] and protein S with anti-TFPI or anti-protein S antibodies; and (ii) in TFPI-depleted or protein S-depleted plasmas supplemented with varying amounts of TFPI[alpha] or protein S. Results Both anti-TFPI and anti-protein S antibodies enhanced thrombin generation in plasma triggered with RVV-X, FXa, FIXa, or FXIa. Anti-TFPI and anti-protein S antibodies decreased the lag time and increased the peak height of thrombin generation to the same extent, indicating that inhibition of FXa by TFPI[alpha] requires the presence of protein S. TFPI[alpha] and protein S titrations in TFPI-depleted or protein S-depleted plasma in which thrombin formation was initiated with triggers other than TF also revealed TF-independent anticoagulant activity of TFPI[alpha], which was completely dependent on the presence of protein S. Conclusion Direct inhibition of FXa by TFPI[alpha] contributes to the downregulation of coagulation.