2015 ACVIM Forum Research Abstract Program

EBDFT placement in dogs is feasible and warrants further investigation to determine if this technique can provide improved outcomes for BES. Thromboembolic events are a common complication of many diseases in veterinary patients, and anti-platelet therapies can be ineffective in their prevention. n-3 polyunsaturated fatty acids (n-3 PUFA) can inhibit platelet function in people, and enhance efficacy of anti-platelet therapy. The objectives of this study were to evaluate the effects of low-dose acetylsalicylic acid (ASA; 1 mg/ kg/day) alone, n-3 PUFA (100 mg/kg/day) alone, and combination ASA and n-3 PUFA therapy on platelet function and activation markers in healthy dogs. Platelet function was measured using platelet function analyzer (PFA) closure time and whole blood aggregometry. Flow cytometry was used to measure platelet activation (P-selectin) and platelet leukocyte aggregates. Above tests were evaluated at baseline, during therapy with ASA and n-3 PUFA alone, and during combination therapy to assess effects on platelet function. This study demonstrated that n-3 PUFA alone did not cause a significant change in platelet function tests. ASA alone decreased platelet function as measured by PFA-epinephrine following one week of therapy (P < 0.0001). ASA plus n-3 PUFA decreased platelet function significantly more versus ASA alone when measured by whole blood aggregometry (agonists: arachidonic acid P = 0.008; collagen P = 0.006). No change in platelet activation was detected via flow cytometry after any therapies. In conclusion, ASA plus n-3 PUFA appear to have a synergistic effect on inhibition of platelet function in healthy dogs. This combination therapy may increase efficacy of anti-platelet therapy in dogs at risk of thromboembolic complications. Neutrophil extracellular traps (NETs) are webs of DNA and proteins that protect against infection, but are pro-thrombotic. Mortality from canine immune-mediated hemolytic anemia (IMHA) largely results from thrombosis, and IMHA is associated with two triggers of NET formation: heme and hypoxia. Therefore, NETs may be prognostic markers or therapeutic targets in IMHA. However, NETs have not been previously demonstrated in dogs. Study aims were (1) to develop a method for in vitro generation of NETs by canine neutrophils and (2) to determine if dogs with primary IMHA have higher circulating nucleosomes (a marker correlated with NETs) than healthy dogs. Neutrophils were isolated from 5 healthy pet dogs and cultured with increasing concentrations of phorbol 12-myristate 13-acetate (PMA) or platelet-activating factor (PAF). Extracellular DNA release was measured using Sytox green fluorescence. Confocal, immunofluorescent (IF) imaging was performed to visualize NETs using DAPI and Cy5 labeled anti-elastase antibodies. Nucleosomes were measured in serum from 20 healthy dogs and 11 IMHA dogs using a commercial ELISA. Four IMHA dogs were excluded due to positive interferences from hemolysis and icterus. Cells stimulated with PAF of ≥31 lM or PMA of ≥0.1 lM released significantly more DNA than unstimulated cells (P < 0.05). DNA release was maximal by 1 hour for PAF and 2 hours for PMA. IF imaging confirmed NET formation. IMHA dogs had significantly higher nucleosomes than controls (median absorbance [arbitrary units] 0.90 and 0.12 respectively, P = 0.01).