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

M Christella L G D Thomassen; Bryan R C Bouwens; Kanin Wichapong; Dennis P Suylen; Freek G Bouwman; Tilman M Hackeng; Rory R Koenen

doi:10.1016/j.jtha.2023.01.017

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

J Thromb Haemost. 2023 May;21(5):1214-1226. doi: 10.1016/j.jtha.2023.01.017. Epub 2023 Jan 28.

Authors

M Christella L G D Thomassen¹, Bryan R C Bouwens¹, Kanin Wichapong¹, Dennis P Suylen¹, Freek G Bouwman², Tilman M Hackeng¹, Rory R Koenen³

Affiliations

¹ Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, the Netherlands.
² Department of Human Biology, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center, Maastricht, the Netherlands.
³ Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, the Netherlands. Electronic address: r.koenen@maastrichtuniversity.nl.

PMID: 36716968
DOI: 10.1016/j.jtha.2023.01.017

Abstract

Background: 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.

Objectives: Here, we show that PAD4 inactivates full-length TFPIα by citrullination of its functional arginines.

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords: blood coagulation; citrullination; extracellular traps; neutrophils; thromboinflammation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Arginine
Factor Xa* / metabolism
Humans
Inflammation
Protein-Arginine Deiminase Type 4
Thrombin* / metabolism

Substances

lipoprotein-associated coagulation inhibitor
Protein-Arginine Deiminase Type 4
Factor Xa
Thrombin
Arginine