OC 76.4 - Citrullination of TFPI impairs its natural anticoagulant activity towards factors Xa, VIIa and tissue factor, and reduces binding of its cofactor protein S.

Wednesday, June 26, 2024

11:00 – 11:15 ICT

Room: 210 A-D

Presenting Author(s)

BB

Bryan RC Bouwens, MSc

PhD candidate
CARIM - department of biochemistry - Maastricht university
Maastricht, Limburg, Netherlands

Co-Author(s)

ST

Stella Thomassen

Scientist
Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University
Maastricht, Limburg, Netherlands
EM

Elke Magdeleyns

Analist
Maastricht University
Maastricht, Limburg, Netherlands
DS

Dennis Suylen

Research technician
Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University
Maastricht, Limburg, Netherlands
Tilman M. Hackeng, PhD

Professor
Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University
Maastricht, Netherlands
Rory R. Koenen

Professor
Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University
Maastricht, Limburg, Netherlands

Background: Neutrophils are known to undergo NETosis by externalization of their DNA and other intracellular contents to entrap and kill invading pathogens. NETosis has been shown to enhance blood coagulation during inflammation. It is known that TFPI binds to these extracellular traps where it may be exposed to the citrullinating enzyme peptidylarginine deiminase 4 (PAD4). Citrullination of TFPI was proved to reduce its effectiveness as a natural anticoagulant towards factor Xa.

Aims: The aim of this work is to study effects of citrullination of TFPI on the inhibition of FXa, FVIIa/TF, the binding of protein S, and the activation of FV.

Methods: The effect of TFPI citrullination on inhibition of FXa and FVIIa/TF was measured by chromogenic assays. Protein S binding was assessed by SPR through covalent coupling of citrullinated TFPI and control TFPI to an SPR chip and perfusing protein S. Variants of the C-terminus of TFPI, including the FV homologous domain, were chemically synthesized by solid phase peptide synthesis with arginine residues exchanged for citrulline residues in all permutations. The influence of the resulting peptides on factor V activation was assessed in both chromogenic and thrombin generation assays.

Results: Citrullination of TFPI resulted in a substantial decrease in its ability to inhibit the FVIIa-TF complex. Exchange of all arginines of the TFPI C-terminal peptide with citrulline fully abolished its capacity to inhibit FV activation. Exchange of single arginines revealed an important role for Arg-274, 285 and 287 in the inhibition of FV activation. SPR analysis revealed that binding to protein S was reduced after citrullination of TFPI.

Conclusion(s): Citrullinated TFPI shows impaired natural anticoagulant roles. In combination with its affinity for negatively charged NETs, TFPI-citrullination is a potential mechanism for thrombus formation during thrombo-inflammation.