Table 1_Non-anticoagulant heparin attenuates histone-mediated platelet–leukocyte aggregation and neutrophil extracellular trap formation in a canine whole blood model.docx

IntroductionSepsis 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.
MethodsEleven 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.
ResultsNACH 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.
ConclusionNACH 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.