Increased levels of NETosis in myeloproliferative neoplasms are not linked to thrombotic events.

Morbidity and mortality of Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) are mainly determined by thromboembolic complications. Thrombus formation is facilitated by a neutrophil-specific form of cell death linked to neutrophil extracellular trap (NET) formation (NETosis). Preclinical and clinical data suggested a potential link between NETosis and thrombosis in MPNs. In this study, we aimed to define the impact of NETosis on clinical end points in a large MPN cohort. NETosis was induced in vitro by ionomycin and quantified by enzyme-linked immunosorbent assay-based nucleosome release assays as well as fluorescent staining of free DNA in samples from 103 MPN patients and 28 healthy donors. NETosis rate was correlated with a broad set of clinical data, such as MPN subtype, mutational status, laboratory variables, history of thrombotic events, and treatment types. Triggered NETosis levels were clearly higher in MPN patients than in healthy donors. Positivity for JAK2 V617F or exon 12 as well as CALR mutations correlate with increased NET formation. However, neither JAK2 allelic burden nor history of thromboembolic complication nor the presence of other risk factors for thrombosis (eg, leukocytosis) were associated with the rate of NETosis. In addition, none of the analyzed laboratory parameters nor the type of treatment significantly impacted the rate of NETosis formation. The biology of MPNs has an impact on NET formation because genetic driver mutations favor induction of NETosis, but this does not seems to translate into important clinical end points such as thromboembolic complications. Therefore, NETosis may play a role in facilitating thrombosis, but it is not a sole causative determinant in MPN-associated thrombophilia.

Comparison of Mitochondrial Incubation Media for Measurement of Respiration and Hydrogen Peroxide Production.

High-Resolution FluoRespirometry is a well-established and versatile approach to study mitochondrial oxygen uptake amperometrically in combination with measurement of fluorescence signals. One of the most frequently applied fluorescent dyes is Amplex UltraRed for monitoring rates of hydrogen peroxide production. Selection of an appropriate mitochondrial respiration medium is of crucial importance, the primary role of which is to support and preserve optimum mitochondrial function. For harmonization of results in a common database, we compared respiration and H2O2 production of permeabilized HEK 293T cells measured in MiR05 (sucrose and K-lactobionate), Buffer Z (K-MES and KCl), MiR07 (combination of MiR05 and Buffer Z), and MiRK03 (KCl). Respiration in a simple substrate-uncoupler-inhibitor titration protocol was identical in MiR05, Buffer Z, and MiR07, whereas oxygen fluxes detected with MiRK03 were consistently lower in all coupling and electron transfer-pathway states. H2O2 production rates were comparable in all four media, while assay sensitivity was comparatively low with MiR05 and MiR07 and higher but declining over time in the other two media. Stability of assay sensitivity over experimental time was highest in MiR05 but slightly less in MiR07. Taken together, MiR05 and Buffer Z yield comparable results on respiration and H2O2 production. Despite the lower sensitivity, MiR05 was selected as the medium of choice for FluoRespirometry due to the highest stability of the sensitivity or calibration constant observed in experiments over periods of up to 2 h.