Citrullination in the C-Terminus of Tissue Factor Pathway Inhibitor-Alpha Impairs the Inhibition of Factor V Activation, and the Synergy with Protein S in Factor Xa Inactivation

Background: The C-terminal region of TFPI hosts a FV homology motif, enabling binding to the acidic region within the B-domain of FV and preventing the activation of FV and its FV-short isoform. This region also assists TFPI in binding with its co-factor, protein S. Truncation of TFPI's C-terminal tail by plasmin, thrombin, or the neutrophil enzyme matrix metalloproteinase-8 leads to less efficient coagulation inhibition. This study investigates whether citrullination of arginine residues within the C-terminal region of TFPI affects coagulation parameters.

Aim: To examine the effects of C-terminal citrullination of TFPI, C-terminal peptides with specific citrullination patterns were investigated on FV activation and in their interactions with protein S.

Methods: We synthesized several variants of the TFPI C-terminal region with arginine replaced by citrulline at different locations (R5, R16, R18, and R22). These variants were assayed for their ability to inhibit prothrombinase activity by preventing FV activation. Their performance was further evaluated using calibrated automated thrombography (CAT) experiments. Additionally, the variants were tested for their interference in the binding between full-length TFPI and protein S and their capacity to inhibit TFPI's anti-FXa activity.

Results: Citrullination at R16 or R18 resulted in a substantial reduction (<50%) in prothrombinase inhibition due to the inability of these peptides to prevent FV activation. Variants with multiple citrullinated residues (R16, R18, and R22) showed severe loss of inhibition (<80%). Complete citrullination (R5, R16, R18, and R22) resulted in complete loss of inhibitory activity towards the prothrombinase complex. In thrombin generation assays, variants with citrulline residues near the FV homology motif impaired thrombin generation inhibition, and fully citrullinated variants failed to inhibit thrombin generation completely. The fully citrullinated variant showed a substantial reduction in competition with full-length TFPI for protein S interactions.

Conclusion: This study demonstrates that citrullination of arginine residues within the C-terminal region of TFPI significantly impairs its ability to inhibit coagulation. Specifically, citrullination at critical sites (R16 and R18) within the FV homology motif results in a substantial reduction in prothrombinase inhibition and thrombin generation suppression. Further compounding citrullination, especially in constructs where R16, R18, and R22 were citrullinated, leads to severe loss of inhibitory function, with complete citrullination rendering TFPI almost entirely ineffective in these roles. Additionally, fully citrullinated TFPI variants show reduced competition with full-length TFPI for protein S interactions, suggesting compromised co-factor binding. These findings indicate that extracellular post-translational modifications such as citrullination can drastically alter the functional integrity of TFPI, highlighting the importance of the C-terminal arginine residues in maintaining its anticoagulant properties. Further research may explore the therapeutic implications of modulating TFPI citrullination in coagulation disorders.

Hackeng:Coagulation Profile B.V: Current holder of stock options in a privately-held company, Other: Founder; Cerus Corporation: Research Funding.