Abstract Number: PB1296
Meeting: ISTH 2022 Congress
Background: Ischemia/reperfusion injury (IRI) is a pathological inflammatory process in which activated infiltrating neutrophils release neutrophil extracellular traps (NETs), a decondensed chromatin meshwork. During NET-formation, the intracellular enzyme peptidyl-arginine deiminase 4 (PAD4) is discharged to the extracellular milieu, where it citrullinates proteins leading to loss of protein function. While NET-derived PAD4 was found to citrullinate plasma proteins in several diseases, its role in IRI is unknown.
Aims: Here, we aim to determine the effect of NETs and PAD4-mediated protein citrullination in a model of acute limb IRI.
Methods: Amputated pig forelimbs were exposed to 1h or 9h of ischemia and then reperfused in vivo for 12h. Limb weight and compartmental pressure were measured, and skeletal muscle was analyzed by immunofluorescence (TUNEL assay and dystrophin staining) to evaluate tissue damage. Myeloperoxidase and citrullinated histone 4 were used to detect neutrophils and NETs, respectively. Candidate citrullinated proteins were immuno-precipitated from plasma samples using an anti-citrulline probe and identified by Western blot.
Results: Limb weight and compartmental pressure were increased (0.3- and 3.4-fold, respectively) in limbs that underwent 9h ischemia compared to 1h. Reperfused limbs after 9h ischemia also displayed higher infiltration of neutrophils and NETs. Interestingly, immunohistochemical analysis revealed that the extent of disruption of skeletal muscle architecture correlated with the presence of NETs, suggesting a role for NETs in tissue damage. Next, we looked for citrullinated plasma fibrinogen and antithrombin, which could play a critical role in IRI. We found a 0.5-fold increase of citrullinated fibrinogen in plasma of pigs which were grafted a limb after 9h ischemia. Citrullinated antithrombin did not rise during IRI.
Conclusion(s): Our data reveal that NETs and levels of citrullinated fibrinogen correlated with tissue damage in IRI. Given its established role in the formation of plasmin-resistant thrombi, this suggests that citrullinated fibrinogen contributes to the thrombo-inflammatory events in IRI.
To cite this abstract in AMA style:Zollet V, Arenas I, Hirsiger S, Brahim B, Petrucci M, Mikulic J, Casoni D, Wang J, Beslac O, Nettelbeck K, Garcia L, Fuest l, Vögelin E, Constantinescu M, Rieben R, Sorvillo N. Elevated NET-Formation and Citrullinated-Fibrinogen are Found in a Porcine Model of Acute Limb Ischemia Reperfusion Injury [abstract]. https://abstracts.isth.org/abstract/elevated-net-formation-and-citrullinated-fibrinogen-are-found-in-a-porcine-model-of-acute-limb-ischemia-reperfusion-injury/. Accessed February 28, 2024.
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