*Article* **Microvesicle Formation Induced by Oxidative Stress in Human Erythrocytes**

**Julia Sudnitsyna 1,**† **, Elisaveta Skverchinskaya 2,**† **, Irina Dobrylko <sup>2</sup> , Elena Nikitina <sup>2</sup> , Stepan Gambaryan 2,3 and Igor Mindukshev 2,\***


Received: 30 August 2020; Accepted: 25 September 2020; Published: 28 September 2020

**Abstract:** Extracellular vesicles (EVs) released by different cell types play an important role in many physiological and pathophysiological processes. In physiological conditions, red blood cell (RBC)-derived EVs compose 4–8% of all circulating EVs, and oxidative stress (OS) as a consequence of different pathophysiological conditions significantly increases the amount of circulated RBC-derived EVs. However, the mechanisms of EV formation are not yet fully defined. To analyze OS-induced EV formation and RBC transformations, we used flow cytometry to evaluate cell esterase activity, caspase-3 activity, and band 3 clustering. Band 3 clustering was additionally analyzed by confocal microscopy. Two original laser diffraction-based approaches were used for the analysis of cell deformability and band 3 activity. Hemoglobin species were characterized spectrophotometrically. We showed that cell viability in *tert*-Butyl hydroperoxide-induced OS directly correlated with oxidant concentration to cell count ratio, and that RBC-derived EVs contained hemoglobin oxidized to hemichrome (HbChr). OS induced caspase-3 activation and band 3 clustering in cells and EVs. Importantly, we showed that OS-induced EV formation is independent of calcium. The presented data indicated that during OS, RBCs eliminated HbChr by vesiculation in order to sacrifice the cell itself, thereby prolonging lifespan and delaying the untimely clearance of in all other respects healthy RBCs.

**Keywords:** erythrocytes; microparticles; oxidative stress; vesiculation; band 3; *tert*-Bytyl hydroperoxide t-BOOH; nitric oxide donor; calcium ionophore A23187
