Tissue factor pathway inhibitor (TFPI) is a slow tight-binding inhibitor that inhibits factor (F)Xa in a biphasic fashion: a rapid formation of loose FXa·TFPI encounter complex is followed by slow rearrangement into a tight FXa·TFPI* complex in which the Kunitz-2 (K2) domain of TFPI binds and inhibits FXa. In the current study, full-length TFPI (TFPIfl) and various truncated TFPI constructs were used to assess the importance of TFPI domains other than K2 in the inhibition of FXa. In the absence of Ca2+ ions, FXa was more effectively inhibited by TFPIfl than Gla-domain less FXa. In turn, Ca2+ ions impaired FXa inhibition by TFPIfl but not by TFPI constructs that lack the C-terminus. This suggests that, in absence of Ca2+ ions, interactions between the C-terminus of TFPI and the Gla-domain of FXa promote FXa-inhibition. TFPIfl and K2K3 had similar efficiencies for encounter complex formation. However, K2K3 showed monophasic inhibition instead of biphasic inhibition, indicating absence of rearrangement into a tight complex. K1K2 and TFPI1-161 showed biphasic inhibition, but had less efficient encounter complex formation than TFPIfl. Finally, K2K3 was a 10-fold more efficient FXa- inhibitor than K2. These results indicate that K3-C-terminus enhances the formation of encounter complex and that K1 is required for isomerisation of the encounter- into tight complex. Since TFPIfl has a 10-fold higher Ki than K2K3-C-terminus, we propose that K1 is not only required for the transition of the loose to the tight FXa·TFPI* complex, but also inhibits FXa·TFPI encounter complex formation. This inhibitory activity is counteracted by K3 and C-terminus.

Tissue factor pathway inhibitor (TFPI) down-regulates the extrinsic coagulation pathway by inhibiting FXa and FVIIa. Both TFPI and FXa interact with several plasma proteins (e. g. prothrombin, FV/FVa, protein S) and non-proteinaceous compounds (e. g. phospholipids, heparin). It was our aim to investigate effects of ligands that bind to FXa and TFPI on FXa inhibition by full-length TFPI (designated TFPI) and truncated TFPI (TFPI1-150). Inhibition of FXa by TFPI and TFPI1-150 and effects of phospholipids, heparin, prothrombin, FV, FVa, and protein S thereon was quantified from progress curves of conversion of the FXa-specific chromogenic substrate CS11-(65). Low concentrations negatively charged phospholipids (~10 µM) already maximally stimulated (up to 5- to 6-fold) FXa inhibition by TFPI. Unfractionated heparin at concentrations (0.2-1 U/ml) enhanced FXa inhibition by TFPI ~8-fold, but impaired inhibition at concentrations > 1 U/ml. Physiological protein S and FV concentrations both enhanced FXa inhibition by TFPI 2- to 3-fold. In contrast, thrombin-activated FV (FVa) impaired the ability of TFPI to inhibit FXa. FXa inhibition by TFPI1-150 was not affected by FV, FVa, protein S, phospholipids and heparin. TFPI potently inhibited FXa-catalysed prothrombin activation in the absence of FVa, but hardly inhibited prothrombin activation in the presence of thrombin-activated FVa. In conclusion, physiological concentrations TFPI (0.25-0.5 nM TFPI) inhibit FXa with a t1/2 between 3-15 minutes. Direct FXa inhibition by TFPI is modulated by physiological concentrations prothrombin, FV, FVa, protein S, phospholipids and heparin indicating the importance of these modulators for the in vivo anticoagulant activity of TFPI.

To cite this article: Peraramelli S, Thomassen S, Heinzmann A, Rosing J, Hackeng TM, Hartmann R, Scheiflinger F, Dockal M. Direct inhibition of factor VIIa by TFPI and TFPI constructs. J Thromb Haemost 2013; 11: 704-14.Summary. Background: Tissue factor pathway inhibitor (TFPI) is a multi-Kunitz domain protease inhibitor that down-regulates the extrinsic coagulation pathway by inhibiting FXa and FVIIa. Objectives: To investigate the role of the three Kunitz domains (KDs) of TFPI in FVIIa inhibition using full-length TFPI (TFPI fl ) and truncated TFPI constructs. Methods: Inhibition of FVIIa with/without relipidated tissue factor (TF) or soluble TF (sTF) by TFPI fl /TFPI constructs was quantified with a FVIIa-specific chromogenic substrate. Results and Conclusions: TFPI fl inhibited TF-FVIIa via a monophasic reaction, which is rather slow at low TFPI fl concentrations (t ½ % 5 min at 2 nM TFPI) and has a K i of 4.6 nM. In the presence of sTF and without TF, TFPI fl was a poor FVIIa inhibitor, with K i values of 122 nM and 1118 nM, respectively. This indicates that phospholipids and TF significantly contribute to FVIIa inhibition by TFPI fl . TFPI constructs without the KD3-c-terminus (TFPI 1-150 and KD1-KD2) were 7-10-fold less effective than TFPI fl in inhibiting TF-FVIIa and sTF-FVIIa, indicating that the KD3-C-terminus significantly contributes to direct inhibition of FVIIa by TFPI. Compared with KD1-KD2, KD1 was a poor TF-FVIIa inhibitor (K i =434 nM), which shows that the KD2 domain of TFPI also contributes to FVIIa inhibition. Protein S stimulated TF-FVIIa inhibition by TFPI fl (K i =0.7 nM). In the presence of FXa, a tight quaternary TFFVIIa-TFPI-FXa complex is formed with TFPI fl , TFPI 1-150 and KD1-KD2, with K i values of < 0.15 nM, 0.5 nM and 0.8 nM, respectively, indicating the KD3-C-terminus is not a prerequisite for quaternary complex formation. Phospholipids and the Gla-domain of FXa are required for quaternary complex formation.

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To cite this article: Peraramelli S, Thomassen S, Heinzmann A, Rosing J, Hackeng TM, Hartmann R, Scheiflinger F, Dockal M. Inhibition of tissue factor:factor VIIa-catalyzed factor IX and factor X activation by TFPI and TFPI constructs. J Thromb Haemost 2014; 12: 1826-37.