Nonassociative immune-mediated hemolytic anemia (nIMHA) is considered the most common autoimmune disease in canine companions and is accountable for significant mortality and financial burden to the pet owners.1–4 The disease is driven by a spontaneous autoimmune response directed against normal glycoprotein molecules on the erythrocyte cell membrane, resulting in erythrocyte destruction by complement-mediated lysis and phagocytosis by the mononuclear phagocyte system.5 As a result, severe acute anemia, prehepatic icterus resulting from the accumulation of unconjugated bilirubin, and a marked systemic inflammatory response are the main clinical features of this disease.1–3,6
Many studies7–10 have demonstrated that dogs with nIMHA are hypercoagulable, and thromboembolism is one of the most common complications.6,11 However, despite being the leading complication and cause of mortality, the pathogenesis of hypercoagulability in canine nIMHA is not well understood. Although pulmonary thromboembolism, due to the release of venous thrombi, is well described in dogs with nIMHA, infarctions involving myocardial, splenic, renal, ileac, and mesenteric arteries have also been reported, suggesting that nIMHA induces thromboembolic events by affecting both platelets and the coagulation cascade.12
Currently, there is no consensus as to the choice of thromboprophylactic therapy in dogs suffering from nIMHA due to the lack of knowledge regarding the specific pathophysiology of thrombus formation.3,7,12,13 Therefore, thromboembolic events are still documented as a prevalent cause of death or euthanasia in dogs with nIMHA, highlighting a crucial need for a better understanding of the underlying pathophysiology of thrombosis of canine nIMHA in order to develop effective thromboprophylactic measures, reduce the incidence of thromboembolic events, and thereby improve the short- and long-term survival of dogs with nIMHA.
Phosphatidylserine (PS) is one of the most abundant phospholipids predominantly located in the inner leaflet of the eukaryotic plasma membrane, including the erythrocyte membrane.14 Redistribution of phospholipids across the inner and outer plasma membranes occurs under various physiological and pathological conditions.14 Previous studies15–17 in human erythrocytes have shown that both oxidative stress and hyperbilirubinemia induce externalization of PS to the outer leaflet of the erythrocyte plasma membrane. Both hyperbilirubinemia and oxidative stress are common findings in dogs with nIMHA as hyperbilirubinemia occurs due to the accumulation of unconjugated bilirubin from released hemoglobin from hemolyzed erythrocytes, whereas oxidative stress results from anemia-induced increased oxygen demands to peripheral tissues, increased nitric oxide production (leads to increased production of pro-oxidant compounds), the high susceptibility of erythrocytes to oxidative injuries, and hemolysis.18–20 The redistribution of PS to the outer leaflet is associated with eryptosis, programmed cell death of erythrocytes, by binding to the PS receptors present on macrophages. This results in phagocytosis of the PS-positive erythrocytes.14,21,22 Externalization of PS is also linked to thrombus formation by the facilitation of prothrombinase activity, which results in the incorporation of erythrocytes into thrombi.12,21 To date, no study has investigated the link between oxidative stress, PS externalization, and thrombus formation in dogs. However, human and canine erythrocytes share a common erythrocyte receptor, P2X7, which causes PS externalization in the presence of ATP.23,24 A previous study25 demonstrated that ATP-induced PS externalization on the erythrocyte membrane is 6 times greater in dogs than in humans. If PS externalization does contribute to thrombus formation, this species difference in sensitivity to ATP could explain why thromboembolic events are more common in dogs with nIMHA than in human hemolytic anemias.
Oxidative stress in inflammatory states, like nIMHA, is commonly described in people. Reactive oxygen species, namely peroxide and hydroxyl radicals, are generated during the immune response through an increase in mitochondrial reactive oxygen species production. For experimental studies, pro-oxidant agents are used in vitro to induce oxidative stress and subsequent cellular alterations.
Two common pro-oxidant agents are tert-butyl hydroperoxide (TBHP) and 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH). Tert-butyl hydroperoxide is a membrane-permanent, water-soluble organic hydroperoxide that causes lipid peroxidation, membrane hyperpolarization, increased reactive oxygen species production, and depletion of intracellular glutathione pool.26–30 2,2′-Azobis(2-amidinopropane) dihydrochloride is a water-soluble azo compound that decomposes at physiological temperature (37 °C) to generate an alkyl radical and is subsequently converted to peroxyl radical to cause cellular oxidative damage under an aerobic environment.31–34 A common antioxidant used in experimental studies in people and dogs is N-acetylcysteine amide (NACA), a lipophilic compound that supplies cysteine, 1 of the 3 components of the tripeptide glutathione, which has potent antioxidant effects through free radical scavenging and reducing capabilities via its associated enzymes.
This study aimed to demonstrate the relationship between oxidative stress and PS externalization in canine erythrocytes in vitro. We hypothesize that oxidative stress induced by TBHP and AAPH will cause PS redistribution to the outer leaflet of the plasma membrane of canine erythrocytes (objective 1) and that exposure to NACA will ameliorate PS externalization (objective 2). Our study provides key preliminary data to justify future research investigating the effect of PS receptor externalization on measures of hypercoagulability in canine nIMHA.
Methods
Study design
This was a prospective, pilot, experimental study using fresh canine erythrocytes collected from a colony of purpose-bred Beagles (n = 6) housed at the Preclinical Research Laboratory of Purdue University College of Veterinary Medicine with approval from the Purdue IACUC (protocol number: 2110002204; approved October 14, 2021). Sample size calculation was performed using the resource equation approach,34,35 and 4 to 6 research dogs were considered appropriate for the current study. Considering the unforeseen circumstances that could result in subject withdrawal during the study, 6 purpose-bred research Beagles were used.
Sample collection
Whole blood (2 mL) was collected from each animal at 3 separate times and placed into EDTA for anticoagulation. Canine erythrocytes were then isolated and resuspended for subsequent experiments. Briefly, erythrocytes were separated from plasma through centrifugation at 600 X g for 5 minutes at 4 °C and washed 3 times with a sodium chloride medium with 0.1% bovine serum albumin (147.5 mM NaCl, 2.5 mM KCl, 5 mM D-glucose, 0.1% bovine serum albumin, and 20 mM HEPES, pH 7.5). The wash was conducted by adding 12 mL of sodium chloride with 0.1% bovine serum albumin and pipetting 3 to 5 times to mix thoroughly with the RBCs. The solution then underwent centrifugation at 600 X g for 5 minutes at 4 °C, and the supernatant was discarded by using the pipette. This procedure was repeated 3 times. Ten microliters of erythrocytes from the pellet were resuspended with the same medium to reach an Hct of 10% for subsequent experiments. Erythrocytes harvested from the first and second blood collections were used for the oxidative challenge experiments (objective 1), and the third blood collection was used for the NACA experiments (objective 2) as described below. The first and second blood sample collections occurred within 7 days for each animal, whereas the third blood sample collection occurred 3 months after the second blood sample collection.
In vitro oxidative challenge (objective 1)
Erythrocytes from each dog were aliquoted to allow exposure to 2 types of oxidative agents, TBHP 70% solution in water (Sigma-Aldrich) and AAPH (Sigma-Aldrich), at various concentrations to induce oxidative stress in vitro (Figure 1). For the TBHP assay, a stock solution of 100 mM TBHP 70% solution in water was made and further diluted with NaCl with 0.1% bovine serum albumin to meet the targeted experimental concentrations (2, 3, and 4 mM). Erythrocytes were exposed to TBHP 70% solution in water at 2, 3, and 4 mM and incubated for 20 minutes at 37 °C with 5% CO2. The dosing and exposure duration were elected based on a previous study26 and preliminary experiments (unpublished data) to prevent harmful cellular effects beyond oxidative stress.36 For the AAPH assay, a stock AAPH solution in NaCl medium (147.5 mM NaCl, 2.5 mM KCl, 5 mM D-glucose, and 20 mM HEPES, pH 7.5) was made at 500 mM and further diluted with NaCl with 0.1% bovine serum albumin to meet the targeted experimental concentrations (30, 40, and 50 mM). Erythrocytes were exposed to AAPH at 30, 40, and 50 mM and incubated for 120 minutes at 37 °C with 5% CO2. The incubation conditions of AAPH were elected based on previous studies30,32,37 on human erythrocytes. Erythrocytes incubated with NaCl with 0.1% bovine serum albumin were used as negative controls, whereas positive controls were incubated with benzoylbenzoyl-ATP (BzATP; Sigma-Aldrich), a P2X7 purinergic receptor agonist that induces PS externalization to the erythrocyte membrane outer leaflet, using previously published methods.24,38 All treatment conditions were run in duplicate. Erythrocyte membrane PS externalization was measured by flow cytometry as described below. The concentrations of oxidative agents used in this experiment were assessed for their capacity to induce PS externalization, and the optimum concentration and agent were used for the following NACA experiment.
In objective 1, canine erythrocytes were isolated from 5 research Beagles and underwent oxidative challenges induced by tert-butyl hydroperoxide (TBHP) or 2,2’-Azobis(2-amidinopropane) dihydrochloride (AAPH). After incubation, erythrocytes were stained with a phosphatidylserine (PS) marker (Annexin V–allophycocyanin [APC]). Flow cytometry was performed to determine the erythrocyte population and their PS externalization (A). In objective 2, the canine erythrocytes were exposed to oxidative stress (3 mM TBHP) with the addition of the potent antioxidant N-acetylcysteine amide (NACA) at 3 different conditions (0, 1, and 3 mM). N-acetylcysteine amide (NACA) without TBHP and DMSO served as negative controls (NEGs), and benzoylbenzoyl-ATP served as the positive control (POS). After incubation, erythrocytes were stained with a PS marker (Annexin V-APC), and PS externalization was measured by flow cytometry (B). The time lapse between the TBHP and AAPH exposure experiments in objective 1 was within 7 days for each dog. The time lapse between objective 1 and objective 2 was 3 months. RBC = Erythrocytes.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.09.0273
N-acetylcysteine amide to ameliorate oxidative stress–induced PS externalization (objective 2)
The isolated erythrocytes from the second blood collection from each dog were aliquoted to allow exposure to oxidative stress with the addition of the potent thiol antioxidant NACA at 3 concentrations (0, 1, and 3 mM). The erythrocytes were exposed to the oxidative agent and various concentrations of NACA simultaneously. Erythrocyte aliquots exposed to BzATP were used as the positive control and NaCl medium as the negative control. Erythrocyte aliquots exposed to NACA at 3 mM and DMSO, the vehicle used to dissolve NACA, were used as additional controls (Figure 1). All treatment conditions were run in duplicate. The intensity of erythrocyte membrane PS externalization was measured according to the following flow cytometry protocol.
Measurement of canine erythrocytes’ PS externalization
After incubation with various experimental conditions stated above, erythrocytes were washed twice and resuspended in PBS with 0.3% bovine serum albumin. Twenty microliters of the erythrocyte suspension were then washed once with 1.5 mL of the Annexin V binding buffer (140 mM NaCl, 5 mM CaCl2, and 10 mM HEPES, pH 7.4). The erythrocyte pellet was resuspended with 96 μL of the Annexin V binding buffer and incubated with 4 μL of allophycocyanin (APC)-conjugated Annexin V (Annexin V APC Ready Flow Conjugate; Thermo Fisher Scientific) for 15 minutes in the dark at room temperature according to manufacturer’s instructions. After incubation, samples were diluted with 400 μL of Annexin V binding buffer and analyzed immediately by flow cytometry (Accuri C6 Flow Cytometer; Becton, Dickinson, and Co). The erythrocyte cloud was identified using the forward and side scatter plot. The height and the area of the forward scatter were plotted, and events were identified along the diagonal axis to avoid doublets. The erythrocytes were gated with 10,000 ± 2,500 events recorded using FlowJo software, version 10 (FlowJo). Phosphatidylserine externalization was detected by positive APC fluorescence at the FL4 channel. The positive gate was defined against untreated erythrocytes in Annexin V binding buffer with and without calcium chloride. The percentage of PS-positive cells and median fluorescence intensity (MFI) were recorded for each sample. Median fluorescence intensity was measured with all erythrocytes combined, including positive and negative PS events.
Statistical analysis
Statistical analyses were conducted using GraphPad Prism, version 9.0 (GraphPad). Median, range, and IQR were used to describe the MFI and percentage of PS-positive events of each sample. Intra-assay coefficients of variability were calculated for duplicated samples. The mean values were obtained from the duplicates of the experimental samples when available. The MFI and percentage of PS-positive events of each sample were log transformed. The log-transformed data were analyzed by the mixed-effects model of 1-way ANOVA to compare between treatment groups. The Tukey post hoc method was used to determine significance within pairs. P < .05 was considered significant.
Results
Animals
Six purpose-bred research Beagles were initially used for the study. All dogs had unremarkable baseline bloodwork (CBC and biochemistry profile) and physical examination findings before the start of the study. The only exception was that 1 dog had an implanted vascular access port from a previous, unrelated study. One dog developed an IM mass located on the left thigh during the study period. Due to the unknown nature of this mass and its possible ability to alter the erythrocyte morphology, function, and metabolism, blood samples collected from this dog were excluded from analyses. Therefore, blood samples from 5 dogs were used for the current study (Figure 1).
Phosphatidylserine externalization of canine erythrocytes exposed to TBHP
Tert-butyl hydroperoxide (70% solution in water) exposure at 3 and 4 mM resulted in significantly higher MFI than the negative control erythrocytes, indicating significant increases in PS externalization on erythrocytes plasma membranes. Tert-butyl hydroperoxide exposure at 2 mM also resulted in a higher MFI compared to the negative control, but it did not reach statistical significance (Figure 2; Supplementary Table S1). Overall, the increased concentration of TBHP resulted in an increase in PS externalization on canine erythrocytes that plateaued at 3 mM as the MFI did not differ significantly between the 3 mM and 4 mM TBHP treatment groups (P = .437). Tert-butyl hydroperoxide exposure at all concentrations resulted in significantly increased percentages of PS-positive events when compared to negative controls (Supplementary Table S1).
Exposure of 3 and 4 mM TBHP resulted in significantly increased PS externalization, expressed as median fluorescence intensity (MFI), on canine erythrocytes compared to the NEG (0 mM). Median fluorescence intensity values were log transformed. *P < .05. ns = Not significant.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.09.0273
Phosphatidylserine externalization of canine erythrocytes exposed to AAPH
Canine erythrocytes exposed to AAPH at 30, 40, and 50 mM resulted in significantly higher MFI as compared to the negative control erythrocytes, indicating significant increases in PS externalization of erythrocytes plasma membranes (Figure 3; Supplementary Table S2). Phosphatidylserine externalization measured by the percentage of PS-positive events demonstrated similar results, with significantly increasing percentages of PS-positive events noted with increasing AAPH concentrations (Supplementary Table S2).
Canine erythrocytes’ PS externalization, expressed as MFI, with different concentrations of AAPH (30, 40, and 50 mM). 2,2’-Azobis(2-amidinopropane) dihydrochloride exposure at all concentrations resulted in significantly higher MFI compared to NEG erythrocytes (0 mM), indicating significant increases in PS externalization on erythrocytes’ plasma membranes. Median fluorescence intensity values were log transformed. **P < .01.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.09.0273
N-acetylcysteine amide ameliorates oxidative stress–induced PS externalization in canine erythrocytes
Based on the results described above, 3 mM TBHP induced an adequate oxidative environment to produce sufficient PS externalization in canine erythrocytes with good contrast compared to the negative controls. Therefore, this oxidative condition was utilized for the NACA experiments. N-acetylcysteine amide at all concentrations demonstrated a protective effect against PS externalization in the canine erythrocytes. The MFI values and percentages of PS-positive events were significantly decreased with each increasing concentration of NACA utilized (Figure 4; Tables 1 and 2). The MFI and percentage of PS-positive events remained low for negative control erythrocytes, erythrocytes incubated with different concentrations of NACA, and erythrocytes incubated with DMSO alone.
Effect of NACA (0 mM, 1 mM, and 3 mM) in preventing PS externalization in canine erythrocytes under oxidative stress (TBHP 3 mM). The degree of PS externalization, expressed as MFI, is not different between all concentrations of NACA and NEG, indicating a protective effect to prevent PS externalization when canine erythrocytes were exposed to oxidative stress. The degree of PS externalization was significantly lower in erythrocytes exposed to oxidative stress and treated with all concentrations of NACA compared to exposure to oxidative stress alone. **P < .01; ****P < .0001.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.09.0273
Median fluorescence intensity (MFI) of phosphatidylserine (PS) externalization in canine erythrocytes exposed to standardized oxidative stress and various concentrations of NACA.
| MFI | |||||
|---|---|---|---|---|---|
| Treatment | Median | Range | IQR | CV (%) | P value |
| 3 mM TBHP, 0 mM NACA | 110,774 | 69,744–144,054 | 90,053–129,114 | 12.20 | — |
| 3 mM TBHP, 1 mM NACA | 799.0 | 368.0–78,993 | 677.0–36,939 | 63.34 | .0028a |
| 3 mM TBHP, 3 mM NACA | 258.0 | 222.0–365.0 | 241.5–281.5 | 5.53 | < .0001a |
| 0 mM TBHP, 0 mM NACA (NEG) | 218.0 | 216.0–240.0 | 216.0–235.5 | — | < .0001a |
| NEG (NACA)b | 215.0 | 210.0–229.0 | 210.0–224.0 | — | < .0001a |
| NEG (DMSO)c | 222.0 | 210.0–233.0 | 214.0–228.0 | — | < .0001a |
| POS (BzATP) | 88797 | 4,596–141,818 | 36,011–123,100 | — | .8298 |
BzATP = Benzoylbenzoyl-ATP. CV = Coefficient of variation. MFI = Median fluorescence intensity. NACA = N-acetylcysteine amide. NEG = Negative control. POS = Positive control. TBHP = Tert-butyl hydroperoxide.
Percentage of positive events of phosphatidyl serine (PS) externalization in canine erythrocytes exposed to standardized oxidative stress and various concentrations of NACA.
| Percentage of positive PS events | |||||
|---|---|---|---|---|---|
| Treatment | Median (%) | Range (%) | IQR (%) | CV (%) | P value |
| 3 mM TBHP, 0 mM NACA | 92.55 | 78.50–95.50 | 79.28–94.45 | 3.66 | — |
| 3 mM TBHP, 1 mM NACA | 39.60 | 30.25–56.70 | 30.45–48.88 | 31.40 | .0009a |
| 3 mM TBHP, 3 mM NACA | 7.22 | 5.83–12.95 | 6.26–12.27 | 24.35 | < .0001a |
| 0 mM TBHP, 0 mM NACA (NEG) | 0.11 | 0.03–0.16 | 0.04–0.15 | — | .0002a |
| NEG (NACA)b | 0.16 | 0.06–0.15 | 0.07–0.14 | — | < .0001a |
| NEG (DMSO)c | 0.09 | 0.13–0.38 | 0.14–0.33 | — | < .0001a |
| POS (BzATP) | 90.10 | 47.40–99.20 | 68.20–98.85 | — | .9951 |
BzATP = Benzoylbenzoyl-ATP. CV = Coefficient of variation. MFI = Median fluorescence intensity. NACA = N-acetylcysteine amide. NEG = Negative control. POS = Positive control. TBHP = Tert-butyl hydroperoxide.
Discussion
The current study investigated the trigger of PS externalization in canine erythrocytes in an in vitro experiment setting. Our study revealed that oxidative stress, a common condition associated with hemolytic and nonhemolytic anemias in humans and dogs,19,39,40 causes PS externalization in canine erythrocytes measured by flow cytometry. The degree of erythrocytes’ PS externalization increased with increasing concentrations of both TBHP and AAPH, with TBHP resulting in more profound erythrocyte PS externalization. Such alteration in erythrocyte membrane PS externalization is also protected by the addition of the thiol antioxidant NACA. These results could serve as the first step to further expand our knowledge in PS and thrombus formation in canine nIMHA.
Despite having a relatively straightforward study design and the use of experimental animals with relatively homogenous traits, the current study still has several limitations. Despite utilizing 2 types of oxidative agents at various concentrations, the study did not perform further objective evaluations of oxidative damage by assessing intracellular glutathione concentration, reactive oxygen species, or degree of lipid peroxidation. Previous studies19,41 have revealed that dogs with hemolytic anemia had higher plasma and lower erythrocyte hemolysate glutathione concentrations compared to healthy dogs, and glutathione peroxidase activity significantly decreased in anemic dogs. Both TBHP and AAPH are oxidative agents known to provoke lipid peroxidation and depletion of the intracellular glutathione pool. Assessments of these markers of oxidative stress in the current study would have been beneficial in providing additional information regarding the cellular injuries and alterations caused by these oxidative agents.
Studies in humans and other animals have shown that PS present on erythrocytes, platelets, and microparticles derived from these 2 cell types facilitate thrombosis.12 However, the current study only measured PS externalization on erythrocytes but not platelets or microparticles, which could underestimate the overall degree of PS externalization and its effect on circulation. The current study also did not use specific surface markers to identify canine erythrocytes. Although anti-human erythrocyte antibody CD235a clone JC159 had been shown to bind to canine erythrocytes in a polymorphic reaction pattern,42 our preliminary experiments (unpublished data) showed that this antibody had little or no detectable binding to erythrocytes harvested from the laboratory Beagles used. Therefore, the erythrocyte gating performed in the current study was accomplished by visual assessment of the scatter plots using forward and side scatter optical detectors.
Additionally, while the NACA did show promising results in preventing PS externalization on canine erythrocyte membranes when exposed to oxidative agents, whether the concentrations used in the current study can be safely achieved clinically is still in question. To our knowledge, there are no in vitro or in vivo studies involving NACA in dogs. Most of the NACA studies to date are in vitro, with a few in vivo studies25,43–46 involving IP administration in murine models. There is 1 pharmacokinetic study47 evaluating NACA and its metabolite in mice receiving oral or IV drug administrations. The study showed that NACA had a 3-to-4-fold-higher capacity in replenishing glutathione compared to NAC administration. The lowest maximum-reached plasma concentration was 138.0 μg/mL with 1 single dose of NACA administered orally at 300 mg/kg. This maximum-reached plasma concentration value would be 0.85 μM with proper unit conversion, which is significantly lower than the concentration used in the current study. Given that drug pharmacokinetics can vary greatly between species and that no experiments using lower NACA concentrations were carried out in the current study, follow-up studies are needed before concluding utilizing NACA as an antioxidant therapy in dogs in a clinical setting. Moreover, NACA is not currently approved by the US FDA to be used in nonexperimental live animals. Therefore, the result of objective 2 from the current study might have little clinical value. Lastly, the erythrocytes treated with NACA, especially at 1 mM, showed a very wide range of the percentage of positive PS events and MFI as well as a high intra-assay coefficient of variance. This could be due to TBHP being a strong and rapid-onset oxidative agent to erythrocytes and that a small time lag between steps of experiments could result in the drastic differences in PS externalization seen. It is also important to note that although NACA significantly improved PS externalization in erythrocytes exposed to TBHP, it did not fully reverse the oxidative damage as the degree of PS externalization was still higher compared to the negative control erythrocytes.
The present study revealed that induced oxidative stress causes PS externalization in canine erythrocytes, and the thiol antioxidant NACA can ameliorate this effect. This leads to a future path to further investigate the significance of PS externalization in canine nIMHA. Particularly, there are very few studies specifically focusing on PS externalization and thrombus formation in canine nIMHA. One study48 showed that immune-mediated hemolytic anemia (IMHA) dogs had increased procoagulant activity associated with PS-bearing microparticles measured by thrombin and factor Xa–generating assays. In this same study mentioned, the number of PS-bearing microparticles was not significantly higher compared to the control group dogs, which could be due to a smaller sample size and a wide spectrum of disease severity. Another study found that most of the IMHA dogs (10 of 11) and precursor-targeted immune-mediated anemia dogs (5 of 6) had increased PS externalization on erythrocyte membranes measured by flow cytometry.38 Recently, RNA sequencing of whole-blood samples from treatment-naïve dogs diagnosed with IMHA revealed that a gene that encodes for phospholipase scramblase, a protein responsible for PS externalization, was highly overexpressed.49 Further endeavors are warranted to explore other triggers of PS externalization in relation to nIMHA (ie, bilirubin), the relationship between PS externalization and thrombus formation in canine nIMHA, and therapeutic applications of NACA or other agents in preventing cellular PS externalization.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
The authors gratefully acknowledge Robyn McCain and the staff at the Preclinical Research Laboratory of Purdue University and Animal Housing Facility for providing care for the research Beagles used in this study.
Disclosures
Dr. Moore is a member of the AJVR Scientific Review Board but was not involved in the editorial evaluation of or decision to accept this article for publication.
No AI-assisted technologies were used in the generation of this manuscript.
Funding
Funding for this study was generously provided by the Immunology Pilot Study Grant from the Veterinary Comparative and Clinical Immunology Society, sponsored by Boehringer Ingelheim.
References
- 1.↑
Swann JW, Skelly BJ. Systematic review of evidence relating to the treatment of immune-mediated hemolytic anemia in dogs. J Vet Intern Med. 2013;27(1):1–9. doi:10.1111/jvim.12028
- 2.
Garden OA, Kidd L, Mexas AM, et al. ACVIM consensus statement on the diagnosis of immune-mediated hemolytic anemia in dogs and cats. J Vet Intern Med. 2019;33(2):313–334. doi:10.1111/jvim.15441
- 3.↑
Swann JW, Garden OA, Fellman CL, et al. ACVIM consensus statement on the treatment of immune-mediated hemolytic anemia in dogs. J Vet Intern Med. 2019;33(3):1141–1172. doi:10.1111/jvim.15463
- 4.↑
Skelly B, Swann J. Canine autoimmune hemolytic anemia: management challenges. Vet Med (Auckl). 2016;7:101–112. doi:10.2147/VMRR.S81869
- 5.↑
Tan E, Bienzle D, Shewen P, Kruth S, Wood D. Potentially antigenic RBC membrane proteins in dogs with primary immune-mediated hemolytic anemia. Vet Clin Pathol. 2012;41(1):45–55. doi:10.1111/j.1939-165X.2011.00391.x
- 6.↑
Goggs R, Dennis SG, Bella AD, et al. Predicting outcome in dogs with primary immune-mediated hemolytic anemia: results of a multicenter case registry. J Vet Intern Med. 2015;29(6):1603–1610. doi:10.1111/jvim.13642
- 7.↑
Morassi A, Bianco D, Park E, Nakamura RK, White GA. Evaluation of the safety and tolerability of rivaroxaban in dogs with presumed primary immune-mediated hemolytic anemia. J Vet Emerg Crit Car. 2016;26(4):488–494. doi:10.1111/vec.12480
- 8.
Goggs R, Wiinberg B, Kjelgaard-Hansen M, Chan DL. Serial assessment of the coagulation status of dogs with immune-mediated haemolytic anaemia using thromboelastography. Vet J. 2012;191(3):347–353. doi:10.1016/j.tvjl.2011.03.015
- 9.
Scott-Moncrieff JC, Treadwell NG, McCullough SM, Brooks MB. Hemostatic abnormalities in dogs with primary immune-mediated hemolytic anemia. J Am Anim Hosp Assoc. 2001;37(3):220–227. doi:10.5326/15473317-37-3-220
- 10.↑
Sinnott VB, Otto CM. Use of thromboelastography in dogs with immune-mediated hemolytic anemia: 39 cases (2000–2008). J Vet Emerg Crit Care. 2009;19(5):484–488. doi:10.1111/j.1476-4431.2009.00455.x
- 11.↑
Carr AP, Panciera DL, Kidd L. Prognostic factors for mortality and thromboembolism in canine immune-mediated hemolytic anemia: a retrospective study of 72 dogs. J Vet Intern Med. 2002;16(5):504–509. doi:10.1111/j.1939-1676.2002.tb02378.x
- 12.↑
Kidd L, Mackman N. Prothrombotic mechanisms and anticoagulant therapy in dogs with immune-mediated hemolytic anemia. J Vet Emerg Crit Car. 2013;23(1):3–13. doi:10.1111/j.1476-4431.2012.00824.x
- 13.↑
Mellett AM, Nakamura RK, Bianco D. A prospective study of clopidogrel therapy in dogs with primary immune-mediated hemolytic anemia. J Vet Intern Med. 2011;25(1):71–75. doi:10.1111/j.1939-1676.2010.0656.x
- 14.↑
Birge RB, Boeltz S, Kumar S, et al. Phosphatidylserine is a global immunosuppressive signal in efferocytosis, infectious disease, and cancer. Cell Death Differ. 2016;23(6):962–978. doi:10.1038/cdd.2016.11
- 15.↑
Nur E, Brandjes DP, Teerlink T, et al. N-acetylcysteine reduces oxidative stress in sickle cell patients. Ann Hematol. 2012;91(7):1097–1105. doi:10.1007/s00277-011-1404-z
- 16.
Amer J, Zelig O, Fibach E. Oxidative status of red blood cells, neutrophils, and platelets in paroxysmal nocturnal hemoglobinuria. Exp Hematol. 2008;36(4):369–377. doi:10.1016/j.exphem.2007.12.003
- 17.↑
Brito MA, Silva RFM, Brites D. Bilirubin induces loss of membrane lipids and exposure of phosphatidylserine in human erythrocytes. Cell Biol Toxicol. 2002;18(3):181–192.
- 18.↑
Maruyama T, Hieda M, Mawatari S, Fujino T. Rheological abnormalities in human erythrocytes subjected to oxidative inflammation. Front Physiol. 2022;13:837926. doi:10.3389/fphys.2022.837926
- 19.↑
Woolcock AD, Serpa PBS, Santos AP, Christian JA, Moore GE. Reactive oxygen species, glutathione, and vitamin E concentrations in dogs with hemolytic or nonhemolytic anemia. J Vet Intern Med. 2020;34(6):2357–2364. doi:10.1111/jvim.15926
- 20.↑
Du VX, Huskens D, Maas C, Dieri RA, Groot PGD, Laat BD. New insights into the role of erythrocytes in thrombus formation. Semin Thromb Hemost. 2014;40(1):72–80. doi:10.1055/s-0033-1363470
- 21.↑
Bigdelou P, Farnoud AM, Program E. Induction of eryptosis in red blood cells using a calcium ionophore. J Vis Exp. (155):10.3791/60659.
- 22.↑
Faulks M, Kuit TA, Sophocleous RA, et al. P2X7 receptor activation causes phosphatidylserine exposure in canine erythrocytes. World J Hematol. 2016;5(4):88. doi:10.5315/wjh.v5.i4.88
- 23.↑
Sluyter R, Shemon AN, Wiley JS. P2X7 receptor activation causes phosphatidylserine exposure in human erythrocytes. Biochem Bioph Res Co. 2007;355(1):169–173. doi:10.1016/j.bbrc.2007.01.124
- 24.↑
Sluyter R, Shemon AN, Hughes WE, et al. Canine erythrocytes express the P2X7 receptor: greatly increased function compared with human erythrocytes. Am J Physiol Regul Integr Comp Physiol. 2007;293(5):2090–2098. doi:10.1152/ajpregu.00166.2007
- 25.↑
Pfaff A, Chernatynskaya A, Vineyard H, Ercal N. Thiol antioxidants protect human lens epithelial (HLE B-3) cells against tert-butyl hydroperoxide-induced oxidative damage and cytotoxicity. Biochem Biophys Rep. 2022;29:101213. doi:10.1016/j.bbrep.2022.101213
- 26.↑
Dremza IK, Lapshina EA, Kujawa J, Zavodnik IB. Oxygen-related processes in red blood cells exposed to tert-butyl hydroperoxide. Redox Rep. 2006;11(4):185–192. doi:10.1179/135100006X116709
- 27.
Roy A, Sil PC. Tertiary butyl hydroperoxide induced oxidative damage in mice erythrocytes: protection by taurine. Pathophysiol. 2012;19(2):137–148. doi:10.1016/j.pathophys.2012.05.001
- 28.
Hierso R, Waltz X, Mora P, et al. Effects of oxidative stress on red blood cell rheology in sickle cell patients. Brit J Haematol. 2014;166(4):601–606. doi:10.1111/bjh.12912
- 29.
Fatemi N, Sanati MH, Zavarehei MJ, et al. Effect of tertiary-butyl hydroperoxide (TBHP)-induced oxidative stress on mice sperm quality and testis histopathology. Andrologia. 2013;45(4):232–239. doi:10.1111/j.1439-0272.2012.01335.x
- 30.↑
Ximenes VF, Lopes MG, Petrônio MS, Regasini LO, Silva DHS, Fonseca LM. Inhibitory effect of gallic acid and its esters on 2,2′-azobis(2-amidinopropane)hydrochloride (AAPH)-induced hemolysis and depletion of intracellular glutathione in erythrocytes. J Agric Food Chem. 2010;58(9):5355–5362. doi:10.1021/jf100233y
- 31.↑
Sagristá ML, García AF, Madariaga MAD, Mora M. Antioxidant and pro-oxidant effect of the thiolic compounds n-acetyl-L-cysteine and glutathione against free radical-induced lipid peroxidation. Free Radic Res. 2009;36(3):329–340. doi:10.1080/10715760290019354
- 32.↑
Yang HL, Korivi M, Lin MK, et al. Antihemolytic and antioxidant properties of pearl powder against 2,2′-azobis(2-amidinopropane) dihydrochloride-induced hemolysis and oxidative damage to erythrocyte membrane lipids and proteins. J Food Drug Anal. 2017;25(4):898–907. doi:10.1016/j.jfda.2016.10.007
- 33.
Maciel E, Neves BM, Santinha D, et al. Detection of phosphatidylserine with a modified polar head group in human keratinocytes exposed to the radical generator AAPH. Arch Biochem Biophys. 2014;548:38–45. doi:10.1016/j.abb.2014.02.002
- 34.↑
Arifin WN, Zahiruddin WM. Sample size calculation in animal studies using resource equation approach. Malays J Med Sci. 2017;24(5):101–105.
- 35.↑
Festing MFW. Guidelines for the design and statistical analysis of experiments in papers submitted to ATLA. Altern Lab Anim. 2001;29(4):427–446. doi:10.1177/026119290102900409
- 36.↑
Koch RE, Hill GE. An assessment of techniques to manipulate oxidative stress in animals. Funct Ecol. 2017;31(1):9–21. doi:10.1111/1365-2435.12664
- 37.↑
Sadowska-Woda I, Sychta B, Rachel M, Bieszczad-Bedrejczuk E. Protective effect of desloratadine against oxidative stress in human erythrocytes in vitro. Environ Toxicol Phar. 2010;30(2):141–146. doi:10.1016/j.etap.2010.05.001
- 38.↑
Lucidi CA, Gerlach JA, Jutkowitz A, Scott MA. Immunoglobulin G and phosphatidylserine in regenerative and nonregenerative immune-mediated anemias of dogs. J Vet Intern Med. 2021;35(6):2713–2721. doi:10.1111/jvim.16278
- 39.↑
Fibach E, Rachmilewitz E. The role of oxidative stress in hemolytic anemia. Curr Mol Med. 2008;8(7):609–619. doi:10.2174/156652408786241384
- 40.↑
Fujii J, Kurahashi T, Konno T, Homma T, Iuchi Y. Oxidative stress as a potential causal factor for autoimmune hemolytic anemia and systemic lupus erythematosus. World J Nephrol. 2014;4(2):213–222. doi:10.5527/wjn.v4.i2.213
- 41.↑
Kendall A, Woolcock A, Brooks A, Moore GE. Glutathione peroxidase activity, plasma total antioxidant capacity, and urinary F2- isoprostanes as markers of oxidative stress in anemic dogs. J Vet Intern Med. 2017;31(6):1700–1707. doi:10.1111/jvim.14847
- 42.↑
Schuberth HJ, Kucinskiene G, Chu RM, Faldyna M. Reactivity of cross-reacting monoclonal antibodies with canine leukocytes, platelets and erythrocytes. Vet Immunol Immunopathol. 2007;119(1–2):47–55. doi:10.1016/j.vetimm.2007.06.013
- 43.↑
Schimel AM, Abraham L, Cox D, et al. N-acetylcysteine amide (NACA) prevents retinal degeneration by up-regulating reduced glutathione production and reversing lipid peroxidation. Am J Pathology. 2011;178(5):2032–2043. doi:10.1016/j.ajpath.2011.01.036
- 44.
Sunitha K, Hemshekhar M, Thushara RM, et al. N-acetylcysteine amide: a derivative to fulfill the promises of n-acetylcysteine. Free Radic Res. 2013;47(5):357–367. doi:10.3109/10715762.2013.781595
- 45.
Bhatti J, Nascimento B, Akhtar U, et al. Systematic review of human and animal studies examining the efficacy and safety of N-acetylcysteine (NAC) and N-acetylcysteine amide (NACA) in traumatic brain injury: impact on neurofunctional outcome and biomarkers of oxidative stress and inflammation. Front Neurol. 2018;8:744. doi:10.3389/fneur.2017.00744
- 46.↑
Wang HJ, Huang YW, Tobwala S, Pfaff A, Aronstam R, Ercal N. The role of N-acetylcysteine amide in defending primary human retinal pigment epithelial cells against tert-butyl hydroperoxide-induced oxidative stress. Free Radic Antioxid. 2017;7(2):172–177. doi:10.5530/fra.2017.2.26
- 47.↑
He R, Zheng W, Ginman T, et al. Pharmacokinetic profile of N-acetylcysteine amide and its main metabolite in mice using new analytical method. Eur J Pharm Sci. 2020;143:105158. doi:10.1016/j.ejps.2019.105158
- 48.↑
Kidd L, Geddings J, Hisada Y, et al. Procoagulant microparticles in dogs with immune-mediated hemolytic anemia. J Vet Intern Med. 2015;29(3):908–916. doi:10.1111/jvim.12583
- 49.↑
Borchert C, Herman A, Roth M, Brooks AC, Friedenberg SG. RNA sequencing of whole blood in dogs with primary immune-mediated hemolytic anemia (IMHA) reveals novel insights into disease pathogenesis. PLoS One. 2020;15(10):e0240975. doi:10.1371/journal.pone.0240975
