Citrullination of TFPI alpha by PAD4 impairs its natural anticoagulant activity towards factors Xa and VIIa/tissue factor, and reduces binding to its cofactor protein S.

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BackgroundNeutrophils are known to externalize their DNA and intracellular contents to neutralize invading pathogens. This process may enhance blood coagulation during inflammation. TFPI binds to extracellular DNA and may be citrullinated by peptidylarginine deiminase 4 (PAD4). Citrullination of TFPI reduces its anticoagulant activity towards factor Xa, but appeared to retain its inhibition of tissue factor (TF)-triggered thrombin generation, indicating a differential regulation of TFPI functions by PAD4. AimThis work aims to study effects of citrullination of TFPI alpha on the inhibition of FXa, FVIIa/TF, and the cofactor activity of protein S. MethodsThe effect of TFPI citrullination on 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 SPR and solid-phase binding assays using immobilized protein S, recombinant TFPI, and synthetic TFPI domains. ResultsCitrullination 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 fourfold. ConclusionCitrullinated 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, as protein S binding to citrullinated TFPI is impaired.