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

OBJECTIVE: 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 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.

APPROACH: Citrullination of TFPIα and of TFPI-constructs by PAD4 was studied by 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 by thrombin generation assays.

RESULTS: Nanomolar PAD4 amounts sufficed to abolish 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 and this causes a loss of factor Xa inhibition. This process might play a role in the increased thrombosis risk associated with inflammation.