Direct inhibition of factor VIIa by TFPI and TFPI constructs. Issue 4 (11th April 2013)

• 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 11:Issue 4(2013)
• Page Start: 704
• Page End: 714
• Publication Date: 2013-04-11
• Subjects: Thrombosis -- Periodicals; Hemostasis -- Periodicals; Blood coagulation disorders -- Periodicals; 616.1
• DOI: 10.1111/jth.12152
• Languages: English
• ISSNs: 1538-7933

Abstract

<abstract abstract-type="main" id="jth12152-abs-0001"> <title>Summary</title> <sec id="jth12152-sec-0001" sec-type="section"> <title>Background</title> <p>Tissue factor pathway inhibitor (TFPI) is a multi‐Kunitz domain protease inhibitor that down‐regulates the extrinsic coagulation pathway by inhibiting FXa and FVIIa.</p> </sec> <sec id="jth12152-sec-0002" sec-type="section"> <title>Objectives</title> <p>To investigate the role of the three Kunitz domains (KDs) of TFPI in FVIIa inhibition using full‐length TFPI (TFPI<sub>fl</sub>) and truncated TFPI constructs.</p> </sec> <sec id="jth12152-sec-0003" sec-type="section"> <title>Methods</title> <p>Inhibition of FVIIa with/without relipidated tissue factor (TF) or soluble TF (sTF) by TFPI<sub>fl</sub>/TFPI constructs was quantified with a FVIIa‐specific chromogenic substrate.</p> </sec> <sec id="jth12152-sec-0004" sec-type="section"> <title>Results and Conclusions</title> <p>TFPI<sub>fl</sub> inhibited TF‐FVIIa via a monophasic reaction, which is rather slow at low TFPI<sub>fl</sub> concentrations (t<sub>½ </sub>≈ 5 min at 2 n<sc>m </sc>TFPI) and has a <italic>K</italic><sub>i</sub> of 4.6 n<sc>m</sc>. In the presence of sTF and without TF, TFPI<sub>fl</sub> was a poor FVIIa inhibitor, with <italic>K</italic><sub>i</sub> values of 122 n<sc>m</sc> and 1118 n<sc>m</sc>, respectively. This indicates that phospholipids and TF significantly contribute to FVIIa inhibition by TFPI<sub>fl</sub>. TFPI constructs without the KD3‐c‐terminus (TFPI<sub>1–150</sub> and KD1‐KD2) were 7–10‐fold less effective than TFPI<sub>fl</sub> 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 (<italic>K</italic><sub>i</sub> =434 n<sc>m</sc>), which shows that the KD2 domain of TFPI also contributes to FVIIa inhibition. Protein S stimulated TF‐FVIIa inhibition by TFPI<sub>fl</sub> (<italic>K</italic><sub>i</sub> =0.7 n<sc>m</sc>). In the presence of FXa, a tight quaternary TF‐FVIIa‐TFPI‐FXa complex is formed with TFPI<sub>fl</sub>, TFPI<sub>1–150</sub> and KD1‐KD2, with <italic>K</italic><sub>i</sub> values of < 0.15 n<sc>m</sc>, 0.5 n<sc>m</sc> and 0.8 n<sc>m</sc>, 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.</p> </sec> </abstract>