Abstract Number: OC 35.3
Meeting: ISTH 2021 Congress
Background: Tissue factor pathway inhibitor (TFPI) is an important regulator of coagulation. Low concentrations are a risk factor for thrombosis. TFPI also is an important link between inflammation and thrombosis. During thrombotic events, TFPI binds to neutrophil extracellular traps (NETs), on which it is proteolytically inactivated by neutrophil elastase. The enzyme protein arginine deiminase 4 (PAD4) is crucial for NET formation, catalyzing citrullination of histones prior to the extrusion of DNA.
Aims: In this study, we show that PAD4 regulates the activity of TFPI by citrullination of its functional arginines.
Methods: Citrullination of TFPI and TFPI-constructs by PAD4 was studied by western blotting and mass spectrometry. Functional consequences were investigated by factor Xa inhibition and by thrombin generation.
Results: Mass spectrometry demonstrated efficient citrullination of full-length TFPI by PAD4. Nanomolar concentrations of PAD4 were sufficient to abolish factor Xa inhibition by TFPI after 50 minutes (figure). Citrullination of full-length TFPI was found to be calcium-ion-, time- and concentration-dependent. Truncated variants K1K2 and TFPI 1-161, and the isolated K2 domain were citrullinated less efficiently by PAD4 than full-length TFPI, implying the involvement of the C-terminus in this process. Negatively charged phospholipids inhibited citrullination, an effect only seen for full-length TFPI. Thrombin generation in TFPI-deficient plasma triggered with tissue factor or Russell’s viper venom-X showed almost complete absent anticoagulant activity of citrullinated TFPI.
Conclusions: To conclude, only full-length TFPI is sensitive to citrullination by PAD4. Citrullinated TFPI loses its ability to inhibit factor Xa. This process might play a role in the increased thrombosis risk associated with inflammation and NET formation.
To cite this abstract in AMA style:Thomassen MCL, Bouwens BR, Wichapong K, Suylen DP, Bouwman FG, Hackeng TM, R Koenen R. Protein Arginine Deiminase 4 Inactivates Tissue Factor Pathway Inhibitor by Post-translational Modification of Functional Arginine Residues [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/protein-arginine-deiminase-4-inactivates-tissue-factor-pathway-inhibitor-by-post-translational-modification-of-functional-arginine-residues/. Accessed October 1, 2023.
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