Citrullination of tissue factor pathway inhibitor alpha by peptidylarginine deiminase 4 impairs its natural anticoagulant activity toward factors Xa and VIIa/tissue factor and reduces binding to its cofactor protein S

Neutrophils are known to externalize their DNA and intracellular contents to neutralize invading pathogens. This process may enhance blood coagulation during inflammation. Tissue factor (TF) pathway inhibitor (TFPI) binds to extracellular DNA and may be citrullinated by peptidylarginine deiminase 4. Citrullination of TFPI reduces its anticoagulant activity toward factor (F)Xa but appears to retain its inhibition of TF-triggered thrombin generation, indicating differential regulation of TFPI functions by peptidylarginine deiminase 4. This work aimed to study the effects of citrullination of TFPI-alpha on the inhibition of FXa, FVIIa/TF, and the cofactor activity of protein S. The effect of TFPI citrullination on the inhibition of FXa and FVIIa/TF was measured by chromogenic assays using purified components and by calibrated automated thrombography. Interaction with protein S was assessed by surface plasmon resonance and solid-phase binding assays using immobilized protein S, recombinant TFPI, and synthetic TFPI domains. Citrullination of TFPI abolished its ability to inhibit FXa- and FXIa-triggered thrombin generation. However, its impaired inhibition of TF-triggered thrombin generation was still enhanced by protein S. Chromogenic assays revealed that citrullinated TFPI was essentially inactive as an inhibitor of the FVIIa-TF complex in the absence of protein S but partially restored by protein S. Interaction studies revealed that binding of citrullinated TFPI to protein S was reduced approximately 4-fold. Citrullinated TFPI shows impaired natural anticoagulant activity. While anti-FXa activity is essentially absent, its anti-TF/FVIIa activity can still be enhanced by protein S. This enhancement is incomplete; however, protein S binding to citrullinated TFPI is impaired.