Characterisation of Citrullinated TFPI and Truncated TFPI Constructs By PAD4 in Model and Plasma Systems

Background: The Kunitz type tissue factor pathway inhibitor (TFPI) is an important regulator in hemostasis. Decreased concentrations are a risk factor for thrombosis and complete TFPI deficiency is associated with lethality. TFPI is also reported to be an important link between inflammation and thrombosis. Neutrophil extracellular traps (NETs) formed by NETosis can bind TFPI which then can be cleaved and inactivated by neutrophil elastase (NE) during thrombotic events. In neutrophils, the enzyme peptide arginine deiminase 4 (PAD4) is central in the citrullination of histones prior to the externalization of DNA during NETosis. In this study, we provide evidence that PAD4 also might regulate the activity of TFPI by posttranslational modification of its functional arginines into citrulline.

Aim: To study the effect of citrullination of TFPI and various TFPI constructs on their functional activity on FXa or thrombin generation.

Methods: Citrullination of TFPI by the neutrophil protein PAD4 was studied in a model system (FXa inhibition) and in plasma system (thrombin generation). Various TFPI constructs, Kunitz (K) domains K1K2, K2, and TFPI1-161 were used to study the effects of citrullination on inhibition of FXa. LC-MS was used to locate the specific sites of citrullination.

Results: This study shows that PAD4 very efficiently citrullinates full lenght TFPI. Very low concentrations of PAD4 were sufficient (K_i 0.4 nM) to abolish FXa inhibition by TFPI. Citrullination is calcium-ion, time- and concentration-dependent. The truncated variants K1K2 and TFPI 1-161 and the isolated K2 domain were citrullinated less efficiently by PAD4 than TFPI, implying the presence of specific binding sites for PAD4 at the C-terminus of TFPI. The presence of phospholipids inhibited the citrullination reaction, an effect only seen for TFPI and not for all the other TFPI variants. Thus, the presence of the C-terminus in TFPI appears to be favourable for citrullination by PAD4.

Thrombin generation in TFPI-deficient plasma triggered with TF or Russell's viper venom (RVV)-X showed almost complete absence of anticoagulant activity of citrullinated TFPI

Conclusions: To conclude, only TFPI is very sensitive to citrullination by PAD4. Citrullinated TFPI has lost its ability to inhibit FXa. This process might play a role in the increased thrombosis risk with inflammation. Further experiments are needed to determine the physiologic or pathologic relevance of this process.