Inhibition of tissue factor:factor VIIa–catalyzed factor IX and factor X activation by TFPI and TFPI constructs. (12th October 2014)

• Record Type: Journal Article
• Authors: Peraramelli, S.; Thomassen, S.; Heinzmann, A.; Rosing, J.; Hackeng, T. M.; Hartmann, R.; Scheiflinger, F.; Dockal, M.
• Is Part Of: Journal of thrombosis and haemostasis. Volume 12:Number 11(2014:Nov.)
• Page Start: 1826
• Page End: 1837
• Publication Date: 2014-10-12
• Subjects: Thrombosis -- Periodicals; Hemostasis -- Periodicals; Blood coagulation disorders -- Periodicals; 616.1
• DOI: 10.1111/jth.12713
• Languages: English
• ISSNs: 1538-7933

Abstract

<abstract abstract-type="main" id="jth12713-abs-0001"> <title>Summary</title> <sec id="jth12713-sec-0001" sec-type="section"> <title>Background</title> <p>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.</p> </sec> <sec id="jth12713-sec-0002" sec-type="section"> <title>Aim</title> <p>To investigate the role of the Kunitz domains of TFPI and protein S in the inhibition of FX and FIX activation.</p> </sec> <sec id="jth12713-sec-0003" sec-type="section"> <title>Methods</title> <p>Inhibition of TF:FVIIa–catalyzed FX and FIX activation by full‐length TFPI (TFPI<sub>FL</sub>) and TFPI constructs was quantified from progress curves of FXa and FIXa generation measured with chromogenic substrates.</p> </sec> <sec id="jth12713-sec-0004" sec-type="section"> <title>Results and conclusions</title> <p>TFPI<sub>FL</sub> inhibited TF:FVIIa–catalyzed FIX activation with a <italic>K</italic><sub>i</sub> of 16.7 nmol L<sup>–1</sup>. Protein S reduced the <italic>K</italic><sub>i</sub> to 1.0 nmol L<sup>–1</sup>. TFPI<sub>1‐150</sub> and KD1‐KD2 had 10‐fold higher <italic>K</italic><sub>i</sub> values and were not stimulated by protein S. Single Kunitz domains were poor inhibitors of TF:FVIIa‐catalyzed FIX activation (K<sub>i</sub> >800 n<sc>m</sc>). FX activation was measured at limiting FVIIa and excess TF or <italic>vice versa</italic>. At both conditions, TFPI<sub>FL</sub>, TFPI<sub>1‐150</sub>, and KD1‐KD2 showed similar inhibition of FX activation. However, at low phospholipid concentrations, TFPI<sub>FL</sub> was ~ 15‐fold more active than TFPI<sub>1‐150</sub> or KD1‐KD2. Apparently, excess phospholipids act as a kind of sink for TFPI<sub>FL</sub>, limiting its availability for TF:FVIIa inhibition. Preformed FXa:TFPI<sub>FL/1‐150</sub> 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.</p> </sec> </abstract>