Protein arginine deiminase 4 inactivates tissue factor pathway inhibitor-alpha by enzymatic modification of functional arginine residues

• Published in: Journal of thrombosis and haemostasis 2023-05, Vol.21 (5), p.1214-1226
• Main Authors: Thomassen, M. Christella L.G.D., Bouwens, Bryan R.C., Wichapong, Kanin, Suylen, Dennis P., Bouwman, Freek G., Hackeng, Tilman M., Koenen, Rory R.
• Format: Article
• Language: English
• Subjects: Arginine; blood coagulation; citrullination; extracellular traps; Factor Xa - metabolism; Humans; Inflammation; neutrophils; Protein-Arginine Deiminase Type 4; Thrombin - metabolism; thromboinflammation
• ISSN: 1538-7836; 1538-7836
• DOI: 10.1016/j.jtha.2023.01.017

Tissue factor pathway inhibitor (TFPI) is an important regulator of coagulation and a link between inflammation and thrombosis. During thrombotic events, TFPI is proteolytically inactivated by neutrophil elastase while bound to neutrophil extracellular traps (NETs). Protein arginine deiminase 4 (PAD4) catalyzes the conversion of arginine to citrulline and is crucial for NET formation. Here, we show that PAD4 inactivates full-length TFPIα by citrullination of its functional arginines. Citrullination of TFPIα and of TFPI-constructs by PAD4 was studied using western blotting and mass spectrometry. Binding of TFPIα to PAD4 was investigated using a solid-phase assay. Functional consequences were investigated by factor Xa inhibition and thrombin generation assays. Nanomolar PAD4 amounts eliminated factor Xa inhibition by TFPIα. A citrullinated mutant Kunitz 2 domain did not inhibit factor Xa. Citrullination of TFPIα was found to be time- and concentration-dependent. Immunoprecipitation of citrullinated proteins from whole blood after neutrophil activation suggested the presence of TFPIα. Negatively charged phospholipids inhibited citrullination and truncated variants K1K2 and TFPI 1-161, and the isolated K2 domain were less efficiently citrullinated by PAD4. TFPIα bound to PAD4 with nanomolar affinity and involved the basic C-terminus. Thrombin generation in TFPI-deficient plasma demonstrated reduced anticoagulant activity of citrullinated TFPI. Mass spectrometry demonstrated citrullination of surface-exposed arginine residues in TFPIα after incubation with PAD4. Full-length TFPIα is sensitive to citrullination by PAD4, which causes loss of factor Xa inhibition. This process may play a role in the increased thrombosis risk associated with inflammation. •Protein arginine deiminase 4 (PAD4) is involved in the formation of neutrophil extracellular traps (NETs); tissue factor pathway inhibitor (TFPI) can bind to NETs.•Coincubation of TFPIα with PAD4 led to loss of TFPI activity and an increase of citrulline residues.•PAD4 can interact with TFPIα. Modification of arginine 107 led to loss of antifactor Xa activity.•Citrullination of TFPIα could contribute to the thrombotic risk associated with inflammatory disease.