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

S. Peraramelli, S. Thomassen, A. Heinzmann, J. Rosing, T. M. Hackeng*, R. Hartmann, F. Scheiflinger, M. Dockal

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

19 Citations (Web of Science)


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. AimTo investigate the role of the Kunitz domains of TFPI and protein S in the inhibition of FX and FIX activation. MethodsInhibition 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 conclusionsTFPI(FL) inhibited TF:FVIIa-catalyzed FIX activation with a K-i of 16.7nmol L-1. Protein S reduced the K-i to 1.0 nmol L-1. TFPI1-150 and KD1-KD2 had 10-fold higher K-i values and were not stimulated by protein S. Single Kunitz domains were poor inhibitors of TF:FVIIa-catalyzed FIX activation (K-i >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.
Original languageEnglish
Pages (from-to)1826-1837
JournalJournal of Thrombosis and Haemostasis
Issue number11
Publication statusPublished - Nov 2014


  • factor IX
  • factor X
  • phospholipids
  • protein S
  • TFPI

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