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Protein arginine deiminase 4 inactivates tissue factor pathway inhibitor-alpha by enzymatic modification of functional arginine residues. 2023

M Christella L G D Thomassen, and Bryan R C Bouwens, and Kanin Wichapong, and Dennis P Suylen, and Freek G Bouwman, and Tilman M Hackeng, and Rory R Koenen
Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, the Netherlands.

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.

UI MeSH Term Description Entries
D007249 Inflammation A pathological process characterized by injury or destruction of tissues caused by a variety of cytologic and chemical reactions. It is usually manifested by typical signs of pain, heat, redness, swelling, and loss of function. Innate Inflammatory Response,Inflammations,Inflammatory Response, Innate,Innate Inflammatory Responses
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000080002 Protein-Arginine Deiminase Type 4 A histone modification enzyme, which converts both ARGININE and monomethyl-arginine to CITRULLINE. It is one of several protein-arginine deiminase isoenzymes. It is a gene regulator involved in APOPTOSIS and CELL DIFFERENTIATION and a potential therapeutic target for the treatment of a variety of diseases. PAD V Enzyme,PADI4 Protein,PADI5 Protein,Peptidyl Arginine Deiminase Type 4,Peptidyl Arginine Deiminase Type IV,Peptidylarginine Deiminase IV,Peptidylarginine Deiminase Type 4,Peptidylarginine Deiminase Type IV,Peptidylarginine Deiminase V,Protein Arginine Deiminase 4,Protein Arginine Deiminase Type 4,Protein Arginine Deiminase Type IV,Protein-Arginine Deiminase Type IV
D001120 Arginine An essential amino acid that is physiologically active in the L-form. Arginine Hydrochloride,Arginine, L-Isomer,DL-Arginine Acetate, Monohydrate,L-Arginine,Arginine, L Isomer,DL Arginine Acetate, Monohydrate,Hydrochloride, Arginine,L Arginine,L-Isomer Arginine,Monohydrate DL-Arginine Acetate
D013917 Thrombin An enzyme formed from PROTHROMBIN that converts FIBRINOGEN to FIBRIN. Thrombase,Thrombin JMI,Thrombin-JMI,Thrombinar,Thrombostat,alpha-Thrombin,beta,gamma-Thrombin,beta-Thrombin,gamma-Thrombin,JMI, Thrombin
D015951 Factor Xa Activated form of factor X that participates in both the intrinsic and extrinsic pathways of blood coagulation. It catalyzes the conversion of prothrombin to thrombin in conjunction with other cofactors. Autoprothrombin C,Coagulation Factor Xa,Factor X, Activated,Thrombokinase,Activated Factor X,Blood Coagulation Factor X, Activated,Factor 10A,Factor Ten A,Factor Xa, Coagulation

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