NETosis promotes cancer-associated arterial microthrombosis presenting as ischemic stroke with troponin elevation

Thålin, Charlotte; Demers, Mélanie; Blomgren, Bo; Wong, Siu Ling; Arbin, Magnus von; Heijne, Anders von; Laska, Ann Charlotte; Wallén, Håkan; Wagner, Denisa D.; Aspberg, Sara

doi:10.1016/j.thromres.2016.01.009

Cited by 137 publications

(159 citation statements)

References 39 publications

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“…This is in line with a recent study revealing a low amount of H3Cit in plasma of healthy individuals when applying a similar assay but without concentrations derived from a standard curve [9]. Furthermore, in accordance with previous studies showing elevations of surrogate markers of NETs in septic patients [10, 11, 25, 26] as well as the detection of H3Cit by western blotting in murine models of LPS-induced septic shock [27, 28], our results support an inflammatory induction of a systemic NET burden by showing clear intra-individual elevations of H3Cit in healthy individuals receiving LPS injection.…”

Section: Discussionsupporting

confidence: 92%

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Validation of an enzyme-linked immunosorbent assay for the quantification of citrullinated histone H3 as a marker for neutrophil extracellular traps in human plasma

et al. 2017

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There is an emerging interest in the diverse functions of neutrophil extracellular traps (NETs) in a variety of disease settings. However, data on circulating NETs rely largely upon surrogate NET markers such as cell-free DNA, nucleosomes, and NET-associated enzymes. Citrullination of histone H3 by peptidyl arginine deiminase 4 (PAD4) is central for NET formation, and citrullinated histone H3 (H3Cit) is considered a NET-specific biomarker. We therefore aimed to optimize and validate a new enzyme-linked immunosorbent assay (ELISA) to quantify the levels of H3Cit in human plasma. A standard curve made of in vitro PAD4-citrullinated histones H3 allows for the quantification of H3Cit in plasma using an anti-histone antibody as capture antibody and an anti-histone H3 citrulline antibody for detection. The assay was evaluated for linearity, stability, specificity, and precision on plasma samples obtained from a human model of inflammation before and after lipopolysaccharide injection. The results revealed linearity and high specificity demonstrated by the inability of detecting non-citrullinated histone H3. Coefficients of variation for intra- and inter-assay variability ranged from 2.1 to 5.1% and from 5.8 to 13.5%, respectively, allowing for a high precision. Furthermore, our results support an inflammatory induction of a systemic NET burden by showing, for the first time, clear intra-individual elevations of plasma H3Cit in a human model of lipopolysaccharide-induced inflammation. Taken together, our work demonstrates the development of a new method for the quantification of H3Cit by ELISA that can reliably be used for the detection of NETs in human plasma.

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Section: Discussionsupporting

confidence: 92%

“…We therefore aimed to validate and optimize an ELISA-based assay recently shown to detect H3Cit in plasma of patients with ischemic stroke [9]. …”

Section: Introductionmentioning

confidence: 99%

Validation of an enzyme-linked immunosorbent assay for the quantification of citrullinated histone H3 as a marker for neutrophil extracellular traps in human plasma

et al. 2017

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“…In summary, we suggest that neutrophils should continue to be explored as therapeutic targets in APS. There is also a need to extend these studies to models beyond venous thrombosis, including arterial thrombosis and thrombosis in the microvasculature (10, 15, 48). …”

Section: Discussionmentioning

confidence: 99%

In Vivo Role of Neutrophil Extracellular Traps in Antiphospholipid Antibody–Mediated Venous Thrombosis

Meng

Yalavarthi

Kanthi

et al. 2017

Arthritis & Rheumatology

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Objective Antiphospholipid syndrome (APS) is a leading acquired cause of thrombotic events. While antiphospholipid antibodies have been shown to promote thrombosis in mice, the role of neutrophils has not been explicitly studied. Here, we characterized neutrophils in the context of a new model of antiphospholipid antibody-mediated venous thrombosis. Methods Mice were administered IgG fractions prepared from patients with APS. At the same time, flow through the inferior vena cava was reduced by a standard stenosis. Resulting thrombi were characterized for size and neutrophil content. Circulating factors and the vessel wall were also assessed. Results As measured by both thrombus weight and thrombosis frequency, mice treated with IgG from patients with APS demonstrated exaggerated thrombosis as compared with controls. APS thrombi were enriched for citrullinated histone H3 (a marker of neutrophil extracellular traps/NETs). APS mice also demonstrated elevated levels of circulating cell-free DNA and human IgG bound to the neutrophil surface. In contrast, circulating neutrophil numbers and markers of vessel wall activation were not appreciably different between APS and control mice. Regarding therapy, treatment with either deoxyribonuclease (which dissolves NETs) or a neutrophil-depleting antibody reduced thrombosis in APS mice to the level seen in controls. Conclusion These data support a mechanism whereby circulating neutrophils are primed by antiphospholipid antibodies to accelerate thrombosis. This line of investigation suggests new, immunomodulatory approaches for the treatment of APS.

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“…This is less common than simply including diagnoses prior to stroke, but the definition varies widely in existing literature [8, 13, 26, 33, 34]. However, based on the results from our initial analyses showing no significant differences among patients diagnosed with cancer pre- and post-stroke, both groups were included as patients with “active cancer”.…”

Section: Discussionmentioning

confidence: 99%

When to Screen Ischaemic Stroke Patients for Cancer

et al. 2018

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Background and Purpose: Ischemic stroke can be the first manifestation of cancer and it is therefore important to ascertain which stroke patients should be considered for cancer-diagnostic investigations. We aimed to determine the frequency of active cancer in patients with acute ischemic stroke and to compare clinical findings in stroke patients with active cancer to ischemic stroke patients with no history of cancer. Finally, we aimed to develop a predictive and feasible score for clinical use to uncover underlying malignancy. Methods: All ischemic stroke patients admitted to the stroke unit in the Department of Neurology, Haukeland University Hospital were consecutively included in the Norwegian Stroke Research Registry (NORSTROKE). Stroke etiology was determined by the Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria. Data on cancer diagnoses was obtained from patients’ medical records and the Cancer Registry of Norway. Active cancer was defined as cancer diagnosis, metastasis of known cancer, recurrent cancer or receiving cancer treatment, all within 12 months before or after the index stroke. Based on variables independently associated with active cancer, a predictive score was developed using the area under the receiver operating characteristic (AUC-ROC) curves. Bayes’ theorem was used to calculate post-test probabilities of active cancer. Results: Of the 1,646 ischemic stroke patients included, 82 (5.0%) had active cancer. Increased D-dimer (OR = 1.1, 95% CI: 1.1–1.2, p = <0.001), lower Hb (OR = 0.6, 95% CI: 0.5–0.7, p = <0.001), smoking (OR = 2.2, 95% CI: 1.2–4.3, p = 0.02) and suffering a stroke of undetermined etiology (OR = 1.9, 95% CI: 1.1–3.3, p = 0.03) were factors independently associated with active cancer. These were included in the final predictive score which gave an AUC of 0.73 (95% CI: 0.65–0.81) in patients younger than 75 years of age. Assuming the prevalence of cancer to be 5%, the score shows that if a patient fulfills all 3 score points, the probability of active cancer is 53%. Conclusions: Active cancer was found in 5% of our ischemic stroke patients. We found that a clinical score comprising elevated D-dimer ≥3 mg/L, lower Hb ≤12.0 g/dL and previous or current smoking is feasible for predicting active cancer in ischemic stroke patients.

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NETosis promotes cancer-associated arterial microthrombosis presenting as ischemic stroke with troponin elevation

Cited by 137 publications

References 39 publications

Validation of an enzyme-linked immunosorbent assay for the quantification of citrullinated histone H3 as a marker for neutrophil extracellular traps in human plasma

Validation of an enzyme-linked immunosorbent assay for the quantification of citrullinated histone H3 as a marker for neutrophil extracellular traps in human plasma

In Vivo Role of Neutrophil Extracellular Traps in Antiphospholipid Antibody–Mediated Venous Thrombosis

When to Screen Ischaemic Stroke Patients for Cancer

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