Non-anticoagulant heparin attenuates histone-mediated platelet-leukocyte aggregation and neutrophil extracellular trap formation in a canine whole blood model.

INTRODUCTION

Sepsis and systemic inflammatory response syndrome (SIRS) are major contributors to morbidity and mortality in dogs, with limited therapeutic advancements. A key factor in this disease progression is the excessive formation of neutrophil extracellular traps (NETs). This study aimed to evaluate the ability of non-anticoagulant heparin (NACH) and unfractionated heparin (UFH) to modulate NET formation and platelet-leukocyte, platelet-neutrophil interactions in an in vitro canine model of septic and aseptic inflammation. We hypothesized that NACH would be non-inferior to UFH in attenuating histone-mediated NET formation and platelet-leukocyte, platelet-neutrophil aggregate formations.

METHODS

Eleven healthy staff-owned dogs were enrolled after confirmation of normal physical examinations and complete blood counts. Whole blood was collected and incubated with either calf thymus histone (0.5 mg/mL) or heated-killed E. coli O111:B4 (1 × 106) to simulate SIRS and sepsis, respectively. Samples were then treated with increasing concentrations of UFH (0, 0.4, 4 and 40 U/mL) or NACH (0, 1, 5, 10 μg/mL). Platelet-leukocyte and neutrophil-platelet aggregates were identified via flow cytometry using fluorophore-conjugated antibodies against platelets (CD61), leukocytes (CD18), and canine neutrophils. NET formation was assessed by quantifying cell-free DNA and intracellular citrullinated histones H3 (citH3) by flow cytometry.

RESULTS

NACH at 10 μg/mL significantly reduced histone-induced platelet-leukocyte (29.0% ± 22.5) and platelet-neutrophil (22.7% ± 15.1) aggregates compared to vehicle controls (37.7% ± 23.7 and 31.8% ± 20.9; p = 0.045). UFH did not significantly reduce histone-induced platelet-leukocyte interactions but showed a dose-dependent reduction in E. coli-induced platelet-leukocyte aggregates, with 40 IU/mL being the most effective (UFH 40 U/mL = 13.65%, IQR: 9.0 to 24.05 vs. UFH 0 U/mL = 18.10%, IQR: 12.68 to 36.18, p = 0.047). Both NACH and UFH modulated neutrophil citH3 expression in E. coli model, but only high-dose NACH was able to module neutrophil extracellular trap formation in the histone mediated model.

CONCLUSION

NACH effectively reduces histone-induced leukocyte and neutrophil-platelet aggregation, while UFH is more effective against E. coli-mediated responses. Both forms of heparin modulate NETs formation, highlighting their distinct stimulus-specific anti-inflammatory effects.