Troponin elevation in acute stroke : clinical characteristics and the link to cancer-associated neutrophil extracellular traps

Elevated plasma levels of troponin, a marker of myocardial injury, are a frequent observation in stroke patients. Despite several reports on an adverse short-term prognosis, however, the significance of troponin elevation in stroke is still controversial, and the myocardial injury often lacks a clear etiology. The aim of this thesis was to determine patient characteristics, including long-term prognosis, in these patients, as well as to explore possible underlying pathomechanisms. In a retrospective cohort analysis of 247 stroke patients (Study I), troponin elevation was significantly associated with age, comorbidity burden, and stroke severity. Stroke patients with troponin elevation also had a higher prevalence of electrocardiographic changes suggestive of myocardial ischemia on admission. A 5-year follow-up period revealed an almost 2-fold increased risk of mortality, with an adjusted hazard ratio of 1.90 (95% CI 1.34- 2.70). In an explorative case-control study (Study II), we furthermore suggest that cancer may be a contributing factor to the poor prognosis in these patients, showing a significant prevalence of underlying cancer among ischemic stroke patients with high troponin elevations. Plasma analyses were strongly supportive of a hypercoagulable state in these patients, and histopathological investigations revealed widespread arterial microthrombi in several organs including the heart. Neutrophil activation, with the release of highly pro-coagulant extracellular chromatin, referred to as neutrophil extracellular traps (NETs), has recently been proposed to play a central role in cancer-associated venous thromboembolism. We therefore proceeded to investigate the role of NETs in the cancer-associated hypercoagulable state seen in the ischemic stroke patients with high levels of plasma troponin as well as an underlying malignancy. As with markers of coagulation, plasma markers of NETs were significantly elevated in these patients, and there were significant positive correlations between the two. Histopathological investigations further supported the role of NETs in the thrombotic state by immunodetection of NET markers in arterial microthrombi. To assess a circulating NET burden in these patients, a novel ELISA-based assay to quantify the NETspecific marker H3Cit in plasma was developed, and subsequently standardized and methodologically validated (Study III) revealing a high specificity, precision and stability of the assay. These results support cardiologic work-up and more aggressive prevention measures in stroke patients with troponin elevation. They furthermore suggest that an underlying cancer should be considered in ischemic stroke patients with unexplainably high plasma levels of troponin. Finally, we link this hypercoagulable state to NETs, and therefore encourage further studies to explore whether markers of NETs could serve as novel diagnostic and prognostic tools in the setting of cancer-associated arterial thrombosis. To this end, we suggest a novel ELISAbased assay to quantify the NET-specific marker H3Cit in plasma

Markers of neutrophil activation and neutrophil extracellular traps in diagnosing patients with acute venous thromboembolism: A feasibility study based on two VTE cohorts

Background Venous thromboembolism (VTE) diagnosis would greatly benefit from the identification of novel biomarkers to complement D-dimer, a marker limited by low specificity. Neutrophil extracellular traps (NETs) have been shown to promote thrombosis and could hypothetically be used for diagnosis of acute VTE. Objectives To assess the levels of specific markers of neutrophil activation and NETs and compare their diagnostic accuracy to D-dimer. Methods We measured plasma levels of neutrophil activation marker neutrophil elastase (NE), the NET marker nucleosomal citrullinated histone H3 (H3Cit-DNA) and cell-free DNA in patients (n = 294) with suspected VTE (pulmonary embolism and deep vein thrombosis) as well as healthy controls (n = 30). A total of 112 VTE positive and 182 VTE negative patients from two prospective cohort studies were included. Results Higher levels of H3Cit-DNA and NE, but not cell-free DNA, were associated with VTE. Area under receiver operating curves (AUC) were 0.90 and 0.93 for D-dimer, 0.65 and 0.68 for NE and 0.60 and 0.67 for H3Cit-DNA in the respective cohorts. Adding NE and H3Cit-DNA to a D-dimer based risk model did not improve AUC. Conclusions Our study demonstrates the presence of neutrophil activation and NET formation in VTE using specific markers. However, the addition of NE or H3Cit-DNA to D-dimer did not improve the discrimination compared to D-dimer alone. This study provides information on the feasibility of using markers of NETs as diagnostic tools in acute VTE. Based on our findings, we believe the potential of these markers are limited in this setting

Patients With COVID-19 Have Elevated Levels of Circulating Extracellular Vesicle Tissue Factor Activity That Is Associated With Severity and Mortality

OBJECTIVE: Patients with coronavirus disease 2019 (COVID-19) have a high rate of thrombosis. We hypothesized that severe acute respiratory syndrome coronavirus 2 leads to induction of TF (tissue factor) expression and increased levels of circulating TF-positive extracellular vesicles (EV) that may drive thrombosis. Approach and Results: We measured levels of plasma EV TF activity in 100 patients with COVID-19 with moderate and severe disease and 28 healthy controls. Levels of EV TF activity were significantly higher in patients with COVID-19 compared with controls. In addition, levels of EV TF activity were associated with disease severity and mortality. Finally, levels of EV TF activity correlated with several plasma markers, including D-dimer, which has been shown to be associated with thrombosis in patients with COVID-19. CONCLUSIONS: Our results indicate that severe acute respiratory syndrome coronavirus 2 infection induces the release of TF-positive EVs into the circulation that are likely to contribute to thrombosis in patients with COVID-19. EV TF activity was also associated with severity and mortality

Neutrophils and neutrophil extracellular traps enhance venous thrombosis in mice bearing human pancreatic tumors

Pancreatic cancer is associated with a high incidence of venous thromboembolism. Neutrophils have been shown to contribute to thrombosis in part by releasing neutrophil extracellular traps (NET). A recent study showed that increased plasma levels of the NET biomarker, citrullinated histone H3 (H3Cit), are associated with venous thromboembolism in patients with pancreatic and lung cancer but not in those with other types of cancer, including breast cancer. In this study, we examined the contribution of neutrophils and NET to venous thrombosis in nude mice bearing human pancreatic tumors. We found that tumor-bearing mice had increased circulating neutrophil counts and levels of granulocyte-colony stimulating factor, neutrophil elastase, H3Cit and cell-free DNA compared with controls. In addition, thrombi from tumor-bearing mice contained increased levels of the neutrophil marker Ly6G, as well as higher levels of H3Cit and cell-free DNA. Thrombi from tumor-bearing mice also had denser fibrin with thinner fibers consistent with increased thrombin generation. Importantly, either neutrophil depletion or administration of DNase I reduced the thrombus size in tumor-bearing but not in control mice. Our results, together with clinical data, suggest that neutrophils and NET contribute to venous thrombosis in patients with pancreatic cancer

Citrullinated histone H3 as a novel prognostic blood marker in patients with advanced cancer

Citrullinated histone H3 (H3Cit) is a central player in the neutrophil release of nuclear chromatin, known as neutrophil extracellular traps (NETs). NETs have been shown to elicit harmful effects on the host, and were recently proposed to promote tumor progression and spread. Here we report significant elevations of plasma H3Cit in patients with advanced cancer compared with age-matched healthy individuals. These elevations were specific to cancer patients as no increase was observed in severely ill and hospitalized patients with a higher non-malignant comorbidity. The analysis of neutrophils from cancer patients showed a higher proportion of neutrophils positive for intracellular H3Cit compared to severely ill patients. Moreover, the presence of plasma H3Cit in cancer patients strongly correlated with neutrophil activation markers neutrophil elastase (NE) and myeloperoxidase (MPO), and the inflammatory cytokines interleukin-6 and -8, known to induce NETosis. In addition, we show that high levels of circulating H3Cit strongly predicted poor clinical outcome in our cohort of cancer patients with a 2-fold increased risk for short-term mortality. Our results also corroborate the association of NE, interleukin-6 and -8 with poor clinical outcome. Taken together, our results are the first to unveil H3Cit as a potential diagnostic and prognostic blood marker associated with an exacerbated inflammatory response in patients with advanced cancer

Circulating H3Cit is elevated in a human model of endotoxemia and can be detected bound to microvesicles.

Early diagnosis of sepsis is crucial since prompt interventions decrease mortality. Citrullinated histone H3 (H3Cit), released from neutrophil extracellular traps (NETs) upon binding of platelets to neutrophils following endotoxin stimulation, has recently been proposed a promising blood biomarker in sepsis. Moreover, microvesicles (MVs), which are released during cell activation and apoptosis and carry a variety of proteins from their parental cells, have also been shown to be elevated in sepsis. In a randomized and placebo-controlled human model of endotoxemia (lipopolysaccharide injection; LPS), we now report significant LPS-induced elevations of circulating H3Cit in 22 healthy individuals. We detected elevations of circulating H3Cit by enzyme-linked immunosorbent assay (ELISA), as well as bound to MVs quantified by flow cytometry. H3Cit-bearing MVs expressed neutrophil and/or platelet surface markers, indicating platelet-neutrophil interactions. In addition, in vitro experiments revealed that H3Cit can bind to phosphatidylserine exposed on platelet derived MVs. Taken together; our results demonstrate that NETs can be detected in peripheral blood during endotoxemia by two distinct H3Cit-specific methods. Furthermore, we propose a previously unrecognized mechanism by which H3Cit may be disseminated throughout the vasculature by the binding to MVs

Neutrophil extracellular trap formation is an independent risk factor for occult cancer in patients presenting with venous thromboembolism

Background: Venous thromboembolism (VTE), particularly unprovoked VTE, is associated with occult cancer. The optimal screening regimen remains controversial. Neutrophil extracellular traps (NETs) are implicated in cancer-associated thrombosis, and elevated biomarkers of NET formation are associated with poor prognosis. Objectives: To investigate the association between NET formation and occult cancer in patients with VTE. Methods: Blood biomarkers associated with NETs and neutrophil activation (nucleo-somal citrullinated histone H3 [H3Cit-DNA], cell-free DNA, and neutrophil elastase) were quantified in patients with VTE. The primary outcome was cancer diagnosed during a one-year follow-up. Results: This study included 460 patients with VTE, of which 221 (48%) had isolated deep vein thrombosis. Forty-three patients had active cancer at inclusion and were excluded from the primary analysis Cancer during follow-up was diagnosed in 29 of 417 (7.0%) patients. After adjustment for age and unprovoked VTE, the hazard ratio of cancer during follow-up per 500 ng/mL increase of H3Cit-DNA was 1.79 (95% CI, 1.03-3.10). Furthermore, patients with cancer-associated VTE (known active cancer or cancer diagnosed during follow-up) had higher levels of H3Cit-DNA than cancer-free patients with VTE after adjustment for age, hemoglobin, gender, chronic obstructive pulmonary disease, previous cancer, and start of anticoagulant treatment (odds ratio 2.06 per 500 ng/mL increase of H3Cit-DNA [95% CI, 1.35-3.13]). Conclusions: H3Cit-DNA is an independent predictor for occult cancer in patients with VTE and elevated in cancer-associated VTE, suggesting that H3Cit-DNA is potentially a useful diagnostic marker for cancer in patients with VTE and that elevated NET formation is a hallmark of cancer-associated VTE

Patients With COVID-19 Have Elevated Levels of Circulating Extracellular Vesicle Tissue Factor Activity That Is Associated With Severity and Mortality

OBJECTIVE: Patients with coronavirus disease 2019 (COVID-19) have a high rate of thrombosis. We hypothesized that severe acute respiratory syndrome coronavirus 2 leads to induction of TF (tissue factor) expression and increased levels of circulating TF-positive extracellular vesicles (EV) that may drive thrombosis. Approach and Results: We measured levels of plasma EV TF activity in 100 patients with COVID-19 with moderate and severe disease and 28 healthy controls. Levels of EV TF activity were significantly higher in patients with COVID-19 compared with controls. In addition, levels of EV TF activity were associated with disease severity and mortality. Finally, levels of EV TF activity correlated with several plasma markers, including D-dimer, which has been shown to be associated with thrombosis in patients with COVID-19. CONCLUSIONS: Our results indicate that severe acute respiratory syndrome coronavirus 2 infection induces the release of TF-positive EVs into the circulation that are likely to contribute to thrombosis in patients with COVID-19. EV TF activity was also associated with severity and mortality

Mucosal immune responses following a fourth SARS-CoV-2 vaccine dose

Supplementary appendixThis appendix formed part of the original submission. We post it as supplied by the authors. Supplement to: Bladh O, Marking U, Havervall S, et al. Mucosal immune responses following a fourth SARS-CoV-2 vaccine dose. Lancet Microbe 2023; published online April 19. https://doi.org/10.1016/S2666-5247(23)00102-7</p

Cancer-associated neutrophil activation correlates with circulating H3Cit and cfDNA.

<p>(<i>A</i>) Peripheral neutrophil count was higher in cancer patients compared to severely ill patients without known cancer (dotted line represents upper reference interval). (<i>B</i>) Flow cytometry analysis showed a higher number of neutrophils positive for intracellular H3Cit in cancer patients compared to both healthy individuals and severely ill patients without known cancer. No significant difference was observed between severely ill patients without known cancer and healthy individuals. (<i>C-D</i>) Plasma NE (<i>C</i>) and MPO (<i>D</i>) were equally elevated in cancer patients and severely ill patients without known cancer compared to healthy individuals. <i>(E)</i> Plasma MPO-DNA complexes were significantly higher in cancer patients compared to both healthy individuals and severely ill patients without known cancer. The levels were also significantly higher in severely ill patients without known cancer compared to healthy individuals. <i>(F)</i> H3Cit and cfDNA correlated positively with NE, MPO and MPO-DNA complexes in cancer patients. (<i>G</i>) Similar correlations were seen in severely ill patients without known cancer, with the exception of correlations between H3Cit and NE and MPO. Lines represent medians with IQR. Groups were compared with the Mann-Whitney U test. Significance of correlation was analyzed with Pearson correlation coefficient after log transformed data to obtain a normal distribution. NS <i>P</i> > 0.05, * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001, **** <i>P</i> < 0.0001.</p