TFPI Expresses Anticoagulant Activity Independent Of TF-FVIIa

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine protease inhibitor that down-regulates the extrinsic coagulation pathway by inhibiting the tissue factor-factor VIIa complex (TF-FVIIa). Inhibition of TF-FVIIa by TFPI is greatly enhanced by FXa. In order to inhibit TF-FVIIa, TFPI first binds to and inhibits Factor Xa (FXa) thus forming a binary TFPI-FXa complex in a reaction that is stimulated by protein S. The TFPI-FXa complex subsequently forms a quaternary complex with TF-FVIIa and blocks the initiation of coagulation. Although TFPI can directly inhibit FXa, this inhibition is generally considered to be of limited physiological significance because the plasma TFPI concentration (0.25-0.5 nM) is several orders of magnitude lower than the plasma concentration of other FXa inhibitors e.g. antithrombin (AT = 2.5-5 μM).

We investigated whether TFPI expresses anticoagulant activity independent of TF-FVIIa i.e. to test whether direct inhibition of FXa by TFPI contributes to the down-regulation of coagulation.

In model systems, the inhibition of FXa by TFPI and AT was determined in the presence of calcium chloride, phospholipids and protein S using a chromogenic substrate to monitor FXa activity. FXa inhibition by TFPI in plasma was determined by measuring thrombin generation curves triggered with FXa, with the FXa activator from Russell’s viper venom (RVV-X) and with FXIa or FIXa in normal pooled plasma. These experiments were performed in the presence of a cocktail of anti TF/anti FVIIa antibodies to inhibit any contribution of traces of TF that might be present in plasma to initiate coagulation. The TF-independent contributions of TFPI and/or protein S to the down-regulation of thrombin generation were quantified 1) after neutralization of TFPI and protein S present in plasma with an anti-TFPI antibody cocktail or anti-protein S antibodies and 2) via TFPI and protein S titrations in TFPI- and in protein S-depleted plasma.

In model systems containing purified proteins, physiological concentrations TFPI (0.25 nM) appeared to be more effective in FXa inhibition (t1/2 = 2.5 min) than physiological concentrations AT (2.5 μM) which inhibited FXa with t1/2 = 3.2 min. Both anti-TFPI and anti-protein S antibodies enhanced thrombin generation that was triggered in plasma with RVV-X, FXa, FIXa or FXIa. Depending on the trigger and trigger concentration used anti-TFPI and anti-protein S antibodies decreased the lag time and/or increased the peak height of thrombin generation. At high trigger concentrations neither anti-TFPI nor anti-protein S antibodies enhanced thrombin generation. TFPI and protein S titrations in TFPI- and protein S-depleted plasma in which thrombin formation was initiated with triggers other than TF confirmed that both TFPI and protein S express TF-independent anticoagulant activity. TFPI titrations in protein S-depleted plasma triggered with RVV-X or FXIa showed that the TF-independent anticoagulant activity of TFPI was greatly reduced in the absence of protein S. Supplementation of the protein S-depleted plasma with protein S restored the anticoagulant effect of TFPI.

We demonstrated that TFPI, despite its low plasma concentration, expresses anticoagulant activity in the absence of TF. Kinetic experiments in model systems at plasma TFPI concentrations show that direct inhibition of FXa by TFPI can account for the TF-independent anticoagulant effect of TFPI on thrombin generation. Blocking the anticoagulant activity of protein S with anti-protein S antibodies also enhances thrombin generation with triggers other than TF. The fact that neutralization of TFPI and of protein S with specific antibodies resulted in a similar stimulation of thrombin generation suggests that TFPI is strongly dependent on the presence of protein S for expression of TF-independent anticoagulant activity in plasma. The ability of TFPI to inhibit thrombin generation initiated via the intrinsic coagulation pathway is an important observation particularly in the light of recent reports which indicate that the intrinsic coagulation pathway plays a relevant role in thrombus formation and in cardiovascular disease.