Inhibition of tissue factor:factor VIIa-catalyzed factor IX and factor X activation by TFPI and TFPI constructs.

BACKGROUND: TFPI is a Kunitz-type protease inhibitor that downregulates the extrinsic coagulation pathway by inhibiting factor Xa (FXa) and FVIIa. All three Kunitz domains (KD1, KD2, and KD3) and protein S are required for optimal inhibition of FXa and FVIIa. There is limited information on Kunitz domain requirements of the inhibition of TF:FVIIa-catalyzed FIX and FX activation by TFPI. AIM: To investigate the role of the Kunitz domains of TFPI and protein S in the inhibition of FX and FIX activation. METHODS: Inhibition of TF:FVIIa-catalyzed FX and FIX activation by full-length TFPI (TFPIFL ) and TFPI constructs was quantified from progress curves of FXa and FIXa generation measured with chromogenic substrates. RESULTS AND CONCLUSIONS: TFPIFL inhibited TF:FVIIa-catalyzed FIX activation with a Ki of 16.7 nmol L(-1) . Protein S reduced the Ki to 1.0 nmol L(-1) . TFPI1-150 and KD1-KD2 had 10-fold higher Ki values and were not stimulated by protein S. Single Kunitz domains were poor inhibitors of TF:FVIIa-catalyzed FIX activation (Ki >800 nm). FX activation was measured at limiting FVIIa and excess TF or vice versa. At both conditions, TFPIFL , TFPI1-150 , and KD1-KD2 showed similar inhibition of FX activation. However, at low phospholipid concentrations, TFPIFL was ~ 15-fold more active than TFPI1-150 or KD1-KD2. Apparently, excess phospholipids act as a kind of sink for TFPIFL , limiting its availability for TF:FVIIa inhibition. Preformed FXa:TFPIFL/1-150 complexes rapidly and stoichiometrically inhibited FIX and FX activation by TF:FVIIa, indicating that binary TFPI:FXa complex formation is the limiting step in TF:FVIIa inhibition. Protein S also enhanced inhibition of TF:FVIIa-catalyzed FX activation by TFPI.

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 (t½  ≈ 5 min at 2 nm TFPI) and has a Ki of 4.6 nm. In the presence of sTF and without TF, TFPIfl was a poor FVIIa inhibitor, with Ki values of 122 nm and 1118 nm, respectively. This indicates that phospholipids and TF significantly contribute to FVIIa inhibition by TFPIfl . TFPI constructs without the KD3-c-terminus (TFPI1-150 and KD1-KD2) were 7-10-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 =434 nm), which shows that the KD2 domain of TFPI also contributes to FVIIa inhibition. Protein S stimulated TF-FVIIa inhibition by TFPIfl (Ki =0.7 nm). In the presence of FXa, a tight quaternary TF-FVIIa-TFPI-FXa complex is formed with TFPIfl , TFPI1-150 and KD1-KD2, with Ki 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.