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Platelet and fibrin clots occlude blood vessels in hemostasis and thrombosis. Here we report a noncanonical mechanism for vascular occlusion based on neutrophil extracellular traps (NETs), DNA fibers released by neutrophils during inflammation. We investigated which host factors control NETs in vivo and found that two deoxyribonucleases (DNases), DNase1 and DNase1-like 3, degraded NETs in circulation during sterile neutrophilia and septicemia. In the absence of both DNases, intravascular NETs formed clots that obstructed blood vessels and caused organ damage. Vascular occlusions in patients with severe bacterial infections were associated with a defect to degrade NETs ex vivo and the formation of intravascular NET clots. DNase1 and DNase1-like 3 are independently expressed and thus provide dual host protection against deleterious effects of intravascular NETs.

Cancer-associated thrombosis often lacks a clear etiology. However, it is linked to a poor prognosis and represents the second-leading cause of death in cancer patients. Recent studies have shown that chromatin released into blood, through the generation of neutrophil extracellular traps (NETs), is procoagulant and prothrombotic. Using a murine model of chronic myelogenous leukemia, we show that malignant and nonmalignant neutrophils are more prone to NET formation. This increased sensitivity toward NET generation is also observed in mammary and lung carcinoma models, suggesting that cancers, through a systemic effect on the host, can induce an increase in peripheral blood neutrophils, which are predisposed to NET formation. In addition, in the late stages of the breast carcinoma model, NETosis occurs concomitant with the appearance of venous thrombi in the lung. Moreover, simulation of a minor systemic infection in tumor-bearing, but not control, mice results in the release of large quantities of chromatin and a prothrombotic state. The increase in neutrophil count and their priming is mediated by granulocyte colony-stimulating factor (G-CSF), which accumulates in the blood of tumor-bearing mice. The prothrombotic state in cancer can be reproduced by treating mice with G-CSF combined with low-dose LPS and leads to thrombocytopenia and microthrombosis. Taken together, our results identify extracellular chromatin released through NET formation as a cause for cancer-associated thrombosis and unveil a target in the effort to decrease the incidence of thrombosis in cancer patients.

A chemical genetics approach identifies the Raf-MEK-ERK signaling system downstream of PKC in formation of the antimicrobial cell structures called neutrophil extracellular traps. The signaling mechanisms leading to the formation of neutrophil extracellular traps (NETs), relevant in infections, sepsis and autoimmune diseases, are poorly understood. Neutrophils are not amenable to studies with conventional genetic techniques. Using a new chemical genetic analysis we show that the Raf-MEK-ERK pathway is involved in NET formation through activation of NADPH oxidase and upregulation of antiapoptotic proteins. We identify potential targets for drugs addressing NET-associated diseases.

Deep vein thrombosis and pulmonary embolism are major health problems associated with high mortality. Recently, DNA-based neutrophil extracellular traps (NETs) resulting from the release of decondensed chromatin, were found to be part of the thrombus scaffold and to promote coagulation. However, the significance of nuclear decondensation and NET generation in thrombosis is largely unknown. To address this, we adopted a stenosis model of deep vein thrombosis and analyzed venous thrombi in peptidylarginine deiminase 4 (PAD4)-deficient mice that cannot citrullinate histones, a process required for chromatin decondensation and NET formation. Intriguingly, less than 10% of PAD4 ⁻/⁻ mice produced a thrombus 48 h after inferior vena cava stenosis whereas 90% of wild-type mice did. Neutrophils were abundantly present in thrombi formed in both groups, whereas extracellular citrullinated histones were seen only in thrombi from wild-type mice. Bone marrow chimera experiments indicated that PAD4 in hematopoietic cells was the source of the prothrombotic effect in deep vein thrombosis. Thrombosis could be rescued by infusion of wild-type neutrophils, suggesting that neutrophil PAD4 was important and sufficient. Endothelial activation and platelet aggregation were normal in PAD4 ⁻/⁻ mice, as was hemostatic potential determined by bleeding time and platelet plug formation after venous injury. Our results show that PAD4-mediated chromatin decondensation in the neutrophil is crucial for pathological venous thrombosis and present neutrophil activation and PAD4 as potential drug targets for deep vein thrombosis.

Neutrophil extracellular traps (NETs) are part of the innate immune response to infections. NETs are a meshwork of DNA fibers comprising histones and antimicrobial proteins. Microbes are immobilized in NETs and encounter a locally high and lethal concentration of effector proteins. Recent studies show that NETs are formed inside the vasculature in infections and noninfectious diseases. Here we report that NETs provide a heretofore unrecognized scaffold and stimulus for thrombus formation. NETs perfused with blood caused platelet adhesion, activation, and aggregation. DNase or the anticoagulant heparin dismantled the NET scaffold and prevented thrombus formation. Stimulation of platelets with purified histones was sufficient for aggregation. NETs recruited red blood cells, promoted fibrin deposition, and induced a red thrombus, such as that found in veins. Markers of extracellular DNA traps were detected in a thrombus and plasma of baboons subjected to deep vein thrombosis, an example of inflammation-enhanced thrombosis. Our observations indicate that NETs are a previously unrecognized link between inflammation and thrombosis and may further explain the epidemiological association of infection with thrombosis.

Neutrophil Extracellular Traps (NETs) are key mediators of immunothrombotic mechanisms and defective clearance of NETs from the circulation underlies an array of thrombotic, inflammatory, infectious, and autoimmune diseases. Efficient NET degradation depends on the combined activity of two distinct DNases, DNase1 and DNase1-like 3 (DNase1L3) that preferentially digest double-stranded DNA (dsDNA) and chromatin, respectively. Here, we engineered a dual-active DNase with combined DNase1 and DNase1L3 activities and characterized the enzyme for its NET degrading potential in vitro. Furthermore, we produced a mouse model with transgenic expression of the dual-active DNase and analyzed body fluids of these animals for DNase1 and DNase 1L3 activities. We systematically substituted 20 amino acid stretches in DNase1 that were not conserved among DNase1 and DNase1L3 with homologous DNase1L3 sequences. We found that the ability of DNase1L3 to degrade chromatin is embedded into three discrete areas of the enzyme's core body, not the C-terminal domain as suggested by the state-of-the-art. Further, combined transfer of the aforementioned areas of DNase1L3 to DNase1 generated a dual-active DNase1 enzyme with additional chromatin degrading activity. The dual-active DNase1 mutant was superior to native DNase1 and DNase1L3 in degrading dsDNA and chromatin, respectively. Transgenic expression of the dual-active DNase1 mutant in hepatocytes of mice lacking endogenous DNases revealed that the engineered enzyme was stable in the circulation, released into serum and filtered to the bile but not into the urine. Therefore, the dual-active DNase1 mutant is a promising tool for neutralization of DNA and NETs with potential therapeutic applications for interference with thromboinflammatory disease states.

Venous thromboembolism (VTE) is a major cause of morbidity and mortality in elderly patients. Extracellular DNA is a pro-inflammatory and pro-thrombotic mediator in vitro and in animal models. Levels of circulating extracellular DNA (ceDNA) are increased in VTE patients, but the association of ceDNA with VTE extent and clinical outcome is poorly understood. We analyzed the association of ceDNA with the extent of VTE, categorized as distal and proximal deep vein thrombosis and pulmonary embolism, and with the clinical outcomes VTE recurrence and mortality. We quantified ceDNA by a fluorescent probe, as well as circulating nucleosomes and neutrophil extracellular traps (NETs) by ELISA in plasma from 611 patients aged ≥ 65 years with acute VTE of a prospective cohort study (SWITCO65+). Levels of ceDNA and nucleosomes, but not NETs, correlated with VTE extent. Infectious comorbidities independently increased ceDNA levels in VTE. CeDNA strongly correlated with C-reactive protein and leukocytosis, suggesting an association of ceDNA with inflammation in VTE patients. CeDNA furthermore predicted PE-related and all-cause mortality, but not VTE recurrence, during a 3-year follow-up. Our study suggests that ceDNA levels in VTE patients reflect the degree of inflammation and may serve as a biomarker to stratify VTE patients at risk for mortality.

Purpose Left ventricular non-compaction (LVNC) is characterized by a 2-layered myocardium composed of a noncompacted (NC) and a compacted (C) layer. The echocardiographic NC:C ratio is difficult to assess in many patients. The aim of the study was to assess the value of cardiac computed tomography (CCT) for the diagnosis of LVNC. Methods In this prospective controlled study, segmental analysis of transthoracic echocardiography (TTE) and prospective ECG-triggered CCT was performed in 17 patients with LVNC and 19 healthy controls. In TTE maximal NC and C thickness was measured at enddiastole and endsystole in the segment with most prominent trabeculation in short axis views. In CCT, maximal segmental NC and C thickness was measured during diastole, and NC:C ratio was determined. Spearman’s correlation coefficient and receiver operating characteristic curves were calculated. Results The median [IQR] radiation dose was 1.3[1.2–1.5]mSv. The CCT thickness of the C layer was significantly lower in patients with LVNC as compared to controls in the inferolateral, midventricular, lateral-, inferior-, and septal-apical segments. The CCT NC:C ratio differed significantly between LVNC and controls in the inferior-midventricular and all the apical segments. NC:C ratio correlated significantly between TTE and CCT at enddiastole (σ = 0.8) and endsystole (σ = 0.9). Using a CCT NC:C ratio ≥1.8, all LVNC patients could be identified. Conclusion LVNC can be diagnosed with ECG-triggered low-dose CCT and discriminated from normal individuals using a NC:C ratio of ≥1.8 in diastole. There is a very good correlation of NC:C ratio in TTE and CCT.

Neutrophil extracellular traps (NETs) are chromatin filaments decorated with enzymes from neutrophil cytoplasmic granules. Anti-neutrophil cytoplasmic antibodies (ANCAs) bind to enzymes from neutrophil cytoplasmic granules and are biomarkers for the diagnosis of systemic vasculitides. ANCA diagnostics are based on indirect immunofluorescence (IIF) of ethanol-fixed neutrophils. IIF shows a cytoplasmic staining pattern (C-ANCA) due to autoantibodies against proteinase 3 (PR3) or a perinuclear staining pattern (P-ANCA) due to autoantibodies against myeloperoxidase (MPO). The distinct ANCA-staining patterns are an artifact of ethanol fixation. Here, we tested NETs as a substrate for the detection of ANCAs in human sera. We observed that P-ANCAs specifically stained NETs, while C-ANCAs targeted the cell bodies of netting neutrophils. The distinct ANCA-staining patterns were caused by the presence of MPO, but not PR3, in NETs. Using NETs as a substrate for IIF, we characterized ANCAs in sera of patients with ANCA-associated vasculitis (AAV). Furthermore, we inhibited serine proteases by diisopropylfluorophosphate to prevent chromatin unfolding and the release of NETs and thus generated neutrophils with MPO-positive nuclei and PR3-positive cytoplasm, which resembled the appearance of ethanol-fixed neutrophils. In conclusion, our data suggest that NETs are selectively loaded with antigens recognized by P-ANCAs, and netting neutrophils provide a physiological substrate for ANCA detection in patients with AAV.

Background Inadequate coronary adenosine response is a potential cause for false negative ischemia testing. Recently, the splenic switch-off (SSO) sign has been identified as a promising tool to ascertain the efficacy of adenosine during vasodilator stress cardiovascular magnetic resonance imaging (CMR). We assessed the value of SSO to predict adenosine response, defined as an increase in myocardial blood flow (MBF) during quantitative stress myocardial perfusion 13 N-ammonia positron emission tomography (PET). Methods We prospectively enrolled 64 patients who underwent simultaneous CMR and PET myocardial perfusion imaging on a hybrid PET/CMR scanner with co-injection of gadolinium based contrast agent (GBCA) and 13N-ammonia during rest and adenosine-induced stress. A myocardial flow reserve (MFR) of  > 1.5 or ischemia as assessed by PET were defined as markers for adequate coronary adenosine response. The presence or absence of SSO was visually assessed. The stress-to-rest intensity ratio (SIR) was calculated as the ratio of stress over rest peak signal intensity for splenic tissue. Additionally, the spleen-to-myocardium ratio, defined as the relative change of spleen to myocardial signal, was calculated for stress (SMR stress ) and rest. Results Sixty-one (95%) patients were coronary adenosine responders, but SSO was absent in 18 (28%) patients. SIR and SMR stress were significantly lower in patients with SSO (SIR: 0.56 ± 0.13 vs. 0.93 ± 0.23; p < 0.001 and SMR stress : 1.09 ± 0.47 vs. 1.68 ± 0.62; p < 0.001). Mean hyperemic and rest MBF were 2.12 ± 0.68 ml/min/g and 0.78 ± 0.26 ml/min/g, respectively. MFR was significantly higher in patients with vs. patients without presence of SSO (3.07 ± 1.03 vs. 2.48 ± 0.96; p = 0.038), but there was only a weak inverse correlation between SMR stress and MFR (R = -0.378; p = 0.02) as well as between SIR and MFR (R = -0.356; p = 0.004). Conclusions The presence of SSO implies adequate coronary adenosine-induced MBF response. Its absence, however, is not a reliable indicator for failed adenosine-induced coronary vasodilatation.