Direct inhibition of factor VIIa by TFPI and TFPI constructs

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 (TFPIfl) and truncated TFPI constructs. Methods Inhibition of FVIIa with/without relipidated tissue factor (TF) or soluble TF (sTF) by TFPIfl/TFPI constructs was quantified with a FVIIa-specific chromogenic substrate. Results and Conclusions TFPIfl inhibited TF-FVIIa via a monophasic reaction, which is rather slow at low TFPIfl concentrations (t1/2 approximate to 5min at 2nm TFPI) and has a Ki of 4.6nm. In the presence of sTF and without TF, TFPIfl was a poor FVIIa inhibitor, with Ki values of 122nm and 1118nm, respectively. This indicates that phospholipids and TF significantly contribute to FVIIa inhibition by TFPIfl. TFPI constructs without the KD3-c-terminus (TFPI1150 and KD1-KD2) were 710-fold less effective than TFPIfl 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 (Ki =434nm), which shows that the KD2 domain of TFPI also contributes to FVIIa inhibition. Protein S stimulated TF-FVIIa inhibition by TFPIfl (Ki =0.7nm). In the presence of FXa, a tight quaternary TF-FVIIa-TFPI-FXa complex is formed with TFPIfl, TFPI1150 and KD1-KD2, with Ki values of <0.15nm, 0.5nm and 0.8nm, 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.