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Inhibition of tissue factor:factor VIIa–catalyzed factor IX and factor X activation by TFPI and TFPI constructs
Author(s) -
Peraramelli S.,
Thomassen S.,
Heinzmann A.,
Rosing J.,
Hackeng T. M.,
Hartmann R.,
Scheiflinger F.,
Dockal M.
Publication year - 2014
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/jth.12713
Subject(s) - chemistry , tissue factor , tissue factor pathway inhibitor , factor x , coagulation , protease , thrombin , thromboplastin , biophysics , protein c , biochemistry , enzyme , platelet , immunology , medicine , biology
Summary Background TFPI is a Kunitz‐type protease inhibitor that downregulates the extrinsic coagulation pathway by inhibiting factor Xa (FXa) and FVII a. All three Kunitz domains ( KD 1, KD 2, and KD 3) and protein S are required for optimal inhibition of FX a and FVII a. There is limited information on Kunitz domain requirements of the inhibition of TF : FVII a–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 : FVII a–catalyzed FX and FIX activation by full‐length TFPI ( TFPI FL ) and TFPI constructs was quantified from progress curves of FX a and FIX a generation measured with chromogenic substrates. Results and conclusions TFPI FL inhibited TF:FVIIa–catalyzed FIX activation with a K i of 16.7 nmol L –1 . Protein S reduced the K i to 1.0 nmol L –1 . TFPI 1‐150 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 n m ). FX activation was measured at limiting FVII a and excess TF or vice versa . At both conditions, TFPI FL , TFPI 1‐150 , and KD 1‐ KD 2 showed similar inhibition of FX activation. However, at low phospholipid concentrations, TFPI FL was ~ 15‐fold more active than TFPI 1‐150 or KD 1‐ KD 2. Apparently, excess phospholipids act as a kind of sink for TFPI FL , limiting its availability for TF : FVII a inhibition. Preformed FX a:TFPI FL/1‐150 complexes rapidly and stoichiometrically inhibited FIX and FX activation by TF : FVII a, indicating that binary TFPI : FX a complex formation is the limiting step in TF : FVII a inhibition. Protein S also enhanced inhibition of TF : FVII a–catalyzed FX activation by TFPI .