Sepsis is the leading cause of critical illness and mortality in human beings and animals. Neutrophils are the primary effector cells of innate immunity during sepsis. Besides degranulation and phagocytosis, neutrophils also release neutrophil extracellular traps (NETs), composed of cell-free DNA, histones, and antimicrobial proteins. Although NETs have protective roles in the initial stages of sepsis, excessive NET formation has been found to induce thrombosis and multiple organ failure in murine sepsis models. Since the discovery of NETs nearly a decade ago, many investigators have identified NETs in various species. However, many questions remain regarding the exact mechanisms and fate of neutrophils following NET formation. In humans and mice, platelet-neutrophil interactions via direct binding or soluble mediators seem to play an important role in mediating NET formation during sepsis. Preliminary data suggest that these interactions may be species dependent. Regardless of these differences, there is increasing evidence in human and veterinary medicine suggesting that NETs play a crucial role in the pathogenesis of intravascular thrombosis and multiple organ failure in sepsis. Because the outcome of sepsis is highly dependent on early recognition and intervention, detection of NETs or NET components can aid in the diagnosis of sepsis in humans and veterinary species. In addition, the use of novel therapies such as deoxyribonuclease and non-anticoagulant heparin to target NET components shows promising results in murine septic models. Much work is needed in translating these NET-targeting therapies to clinical practice.

Objectives:To systematically review available evidence to determine when small animals at risk of thrombosis should be treated with antiplatelet agents and anticoagulants, which antiplatelet and anticoagulant agents are most effective, and when multimodal therapy is indicated.Design: Standardized, systematic evaluation of the literature, categorization of relevant articles according to level of evidence (LOE) and quality (Good, Fair, or Poor), and development of consensus on conclusions via a Delphi-style survey for application of the concepts to clinical practice.Draft recommendations were presented at 2 international veterinary conferences and made available for community assessment, review, and comment prior to final revisions and publication.Settings: Academic and referral veterinary medical centers. Results: Databases searched included Medline via PubMed and CAB abstracts. Twelve PopulationIntervention Comparison Outcome questions were devised and generated corresponding worksheets investigating indications for use of antithrombotic drugs in small animals. Seventy-eight studies were reviewed in detail. Most studies assessed were experimentally controlled laboratory studies in companion animals (56 LOE 3) with smaller numbers of LOE 2 (1), LOE 4 (5), LOE 5 (6), and LOE 6 (4) studies assessed. Only 5 randomized controlled clinical trials were identified (LOE 1, Good-Fair). The 12 worksheets generated 21 guidelines with 17 guideline statements that were refined during 3 rounds of Delphi surveys. A high degree of consensus was reached across all guideline recommendations during the Delphi process. Conclusions:Overall, systematic evidence evaluations generated 2 strong recommendations, 19 weak recommendations (formulated as suggestions), 9 situations where the evidence was insufficient to make strong recommendations, and 8 situations where no relevant evidence was retrieved to aid guideline generation. Numerous significant knowledge gaps were highlighted by the evidence reviews undertaken, indicating the need for substantial additional research in this field.

Immunothrombosis is a potentially beneficial physiological process that aids innate immunity and host defense against pathogen invasion. However, this process can also be damaging when it occurs to excess or in critical blood vessels. Formation of extracellular traps by leukocytes, particularly neutrophils, is central to our understanding of immunothrombosis. In addition to degranulation and phagocytosis, extracellular traps are the third mechanism by which neutrophils combat potential pathogens. These traps consist of extracellular DNA decorated with bactericidal cellular proteins, including elastase, myeloperoxidase, and cathepsins. Neutrophils can release these structures as part of a controlled cell-death process or via a process termed vital NETosis that enables the cells to extrude DNA but remain viable. There is accumulating evidence that NETosis occurs in companion animals, including dogs, horses, and cats, and that it actively contributes to pathogenesis. Numerous studies have been published detailing various methods for identification and quantification of extracellular trap formation, including cell-free DNA, measurements of histones and proteins such as high-mobility group box–1, and techniques involving microscopy and flow cytometry. Here, we outline the present understanding of these phenomena and the mechanisms of extracellular trap formation. We critically review the data regarding measurement of NETosis in companion animals, summarize the existing literature on NETosis in veterinary species, and speculate on what therapeutic options these insights might present to clinicians in the future.

BackgroundClopidogrel is commonly prescribed to cats with perceived increased risk of thromboembolic events, but little information exists regarding its antiplatelet effects.ObjectiveTo determine effects of clopidogrel on platelet responsiveness in cats with or without the A31P mutation in the MYBPC3 gene. A secondary aim was to characterize variability in feline platelet responses to clopidogrel.AnimalsFourteen healthy cats from a Maine Coon/outbred mixed Domestic cat colony: 8 cats homozygous for A31P mutation in the MYPBC3 gene and 6 wild‐type cats without the A31P mutation.MethodsEx vivo study. All cats received clopidogrel (18.75 mg PO q24h) for 14 days. Before and after clopidogrel treatment, adenosine diphosphate (ADP)‐induced P‐selectin expression was evaluated. ADP‐ and thrombin‐induced platelet aggregation was measured by optical aggregometry (OA). Platelet pVASP and ADP receptor response index (ARRI) were measured by Western blot analysis.ResultsPlatelet activation from cats with the A31P mutation was significantly (P = .0095) increased [35.55% (18.58–48.55) to 58.90% (24.85–69.90)], in response to ADP. Clopidogrel treatment attenuated ADP‐induced P‐selectin expression and platelet aggregation. ADP‐ and PGE 1‐treated platelets had a similar level of pVASP as PGE 1‐treated platelets after clopidogrel treatment. Clopidogrel administration resulted in significantly lower ARRI [24.13% (12.46–35.50) to 11.30% (−7.383 to 23.27)] (P = .017). Two of 13 cats were nonresponders based on OA and flow cytometry.Conclusion and Clinical ImportanceClopidogrel is effective at attenuating platelet activation and aggregation in some cats. Cats with A31P mutation had increased platelet activation relative to the variable response seen in wild‐type cats.

Neutrophils release neutrophil extracellular traps (NETs), which are extracellular chromatin decorated with histones and antimicrobial proteins. Although known for antimicrobial properties, overzealous production of NETs (NETosis) may lead to cytotoxicity and multiple organ failure in sepsis. Pathogen-induced NETosis has been extensively studied in mice but its importance in dogs remains largely unknown. This study sought to characterize in vitro NETosis induced by E.coli LPS, including assessing the role of peptidylarginine deiminase (PAD) in canine NETosis. Neutrophils (1×10 cells/ml) from healthy dogs were isolated and treated with 100μg/ml LPS, 100nM phorbol 12-myristate 13-acetate (PMA), or buffer for either 90 or 180min. NETs were assessed using fluorescence microscopy of living neutrophils and immunofluorescent microscopy. Supernatant and cellular debris were purified and cell-free DNA was quantified by spectrophotometry. The role of PAD was assessed by treating LPS- and PMA-activated neutrophils with 50, 100 or 200μM of the PAD inhibitor, Cl-amidine. In vitro NETosis was characterized by co-localization of cell-free DNA, citrullinated histone H3, and myeloperoxidase. LPS stimulation resulted in intracellular citrullination of histone H3. Compared to PMA chemically-induced NETosis, LPS resulted in smaller NETs with less extracellular citrullinated histone H3. Cl-amidine decreased citrullination of histones and NET production in either LPS- or PMA-stimulated neutrophils demonstrating that neutrophil PAD is essential for these cellular processes.