Cl-amidine attenuates neutrophil extracellular trap-enclosed extracellular vesicle (NET-EV)-mediated thrombosis in diabetic mice.

BACKGROUND

Neutrophils can eject out neutrophil extracellular trap (NET)-enclosed extracellular vesicle (NET-EVs) in response to stimuli such as Staphylococcus aureus. Though enhanced thrombosis associated with NET is documented, the thrombotic potential of NET-EVs is not known.

AIM: After confirming the presence of NET-EVs in vivo in diabetes-induced BALB/c mouse, this study analysed thrombosis associated with NET-EV release.

METHODS

Two sets of experiments were designed to check the procoagulant activity connected to the release of NET-EVs. In the first set, to the diabetic mice, either aspirin, Cl-amidine or aspirin + Cl-amidine were administered orally. Bleeding time, spontaneous platelet aggregation, and clot lysis were analysed after 10 days. Histopathological analyses of heart, liver, lung, and kidney tissues were also performed. As Cl-amidine increased bleeding time effectively, it was used for a second experiment. Here, diabetic mice were triggered by an intraperitoneal injection of Staphylococcus aureus culture supernatant (SCS) to trigger NET formation. Presence of NET-EV was confirmed in them using anti-citrullinated histone-H4 and anti-CD63. Cl-amidine was given to one group of these mice and all the tests were performed as before.

RESULTS

Citrullinated histone-H4 in the cells and NET-EV was reduced after Cl-amidine treatment. Both Cl-amidine and aspirin increased bleeding time. Cl-amidine reduced spontaneous platelet aggregation and enhanced clot lysis more efficiently even after SCS-treatment. While no tissue alteration was seen in the Cl-amidine-treated mice, aspirin-treated mice showed haemorrhage and oedema in the heart and lung tissues.

CONCLUSION

Cl-amidine showed the ability to reduce procoagulant activity associated with NET-EV release.