Citrullination of TFPI By PAD4 Impairs Its Natural Anticoagulant Activity Towards Factors Xa, VIIa and Tissue Factor, and Reduces Binding to Its Cofactor Protein S

Background: Neutrophils are known to externalize their DNA and other intracellular contents to entrap and kill invading pathogens. This process has been shown to enhance blood coagulation during inflammation. Tissue Factor Pathway Inhibitor (TFPI) is a natural anticoagulant that inhibits coagulation factors Xa and VIIa/TF, playing a critical role in the regulation of the extrinsic pathway of coagulation. TFPI binds to DNA released by neutrophils, where it may be exposed to the citrullinating enzyme peptidylarginine deiminase 4 (PAD4). Citrullination of TFPI reduces its effectiveness as a natural anticoagulant towards factor Xa but appears to retain part of its inhibition of tissue factor (TF)-triggered thrombin generation, indicating a differential regulation of TFPI functions by PAD4.

Aim: This study aims to investigate the effects of citrullination of TFPI on the inhibition of FXa, FVIIa/TF, and the cofactor activity of protein S.

Methods: The effect of TFPI citrullination on inhibition of FXa and FVIIa/TF was measured by chromogenic assays using purified components and by calibrated automated thrombography (CAT). The interaction with protein S was assessed by surface plasmon resonance (SPR) and solid-phase binding assays using immobilized protein S, recombinant TFPI, and synthetic TFPI domains. Thrombin generation was monitored using normal pooled plasma initiated with FXa, FXIa, and TF.

Results: Citrullination of TFPI abolished its ability to inhibit FXa- and FXIa-triggered thrombin generation. However, its impaired inhibition of TF-triggered thrombin generation could be partially rescued 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. Additionally, thrombin generation assays in protein S- and TFPI-depleted plasma showed that protein S could partially restore the anticoagulant activity of citrullinated TFPI in TF-triggered thrombin generation.

Conclusion: Citrullinated TFPI exhibits impaired natural anticoagulant activity, significantly losing its anti-FXa function while retaining some capacity to inhibit TF-triggered thrombin generation, which can be partially rescued by protein S. Despite the partial rescue, the interaction between citrullinated TFPI and protein S is notably weakened. This study underscores the importance of the interaction between TFPI and protein S in maintaining hemostasis and reveals the potential impact of PAD4-mediated citrullination on coagulation regulation during inflammation. The findings suggest that therapies targeting citrullination or enhancing TFPI-protein S interaction could be beneficial in inflammatory conditions associated with increased thrombotic risk.