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

Abstract

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.