Neutrophil extracellular traps in canine immune-mediated hemolytic anemia

Abstract

<p>Approximately 30% of dogs affected by immune-mediated hemolytic anemia (IMHA) die or are euthanized, often due to thrombosis. Neutrophils may contribute directly to thrombosis by releasing strands of DNA associated with nuclear and extra-nuclear proteins. These structures, termed neutrophil extracellular traps (NETs), have pro-thrombotic properties in humans and mice. This thesis addresses three major questions: (1) do canine neutrophils produce NETs? (2) do NETs activate the canine coagulation system? and (3) is cell-free DNA, a marker correlated with NET formation, elevated in dogs with spontaneous IMHA?</p> <p>Canine granulocytes were incubated with platelet activating factor or phorbol 12-myristate 13-acetate. Both agonists increased fluorescence of Sytox green, a nucleic acid dye incapable of entering viable cells. Fluorescent imaging for DNA and elastase confirmed NET release.</p> <p>A spectrophotometric assay was developed to measure canine clot formation and lysis. This did detect altered clot kinetics in dogs with various disorders, but was unsuitable for use in IMHA due to interferences from hemoglobin and bilirubin. The assay did however provide the basis for in vitro investigations of the effect of NETs on coagulation. When NETs were added to citrated plasma, maximum clot formation velocity was increased and time to 50% clot lysis delayed. The effect on clot lysis but not formation velocity was reduced by DNase digestion, suggesting the latter was mediated by NET proteins. However, at higher concentrations, DNA alone could also increase maximum clot formation velocity. By flow cytometry, NETs were shown to increase phosphatidylserine and P-selectin exposure on canine platelets. This persisted after DNase digestion. Therefore, intact NETs, NET proteins and DNA have pro-thrombotic properties.</p> <p>Plasma was collected from 28 dogs with spontaneous IMHA and 20 healthy controls. Cell-free DNA was purified and measured by PicoGreen fluorescence. Cell-free DNA was increased and as DNase activity was not reduced in IMHA, this was attributed to increased release rather than impaired degradation of DNA. Potential sources of DNA include NETs, necrosis and apoptosis.</p> <p>In conclusion, canine neutrophils release NETs and both NETs and DNA alone promote hypercoagulability. Cell-free DNA is increased in IMHA, suggesting NETs or other forms of cell death may contribute to thrombosis.</p>