The Use of Fluorescently Labeled ARC1779 Aptamer for Assessing the Effect of H₂O₂ on von Willebrand Factor Exocytosis

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Piotr. P. Avdonin¹, Sergey K. Trufanov¹, Elena Yu. Rybakova¹, Aleksandra A. Tsitrina¹, Nikolay V. Goncharov^2,3, and Pavel V. Avdonin^1,a*

¹Koltsov Institute of Developmental Biology, Russian Academy of Sciences, 119334 Moscow, Russia

²Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, 194223 St. Petersburg, Russia

³Research Institute of Hygiene, Occupational Pathology and Human Ecology, Federal Medical-Biological Agency, 188663 Kuzmolovsky, Leningrad Region, Russia

^* To whom correspondence should be addressed.

Received June 5, 2020; Revised October 15, 2020; Accepted October 15, 2020

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Here, we propose a new approach for quantitative estimation of von Willebrand factor (vWF) exposed on the surface of endothelial cells (ECs) using the ARC1779 aptamer that interacts with the vWF A1 domain. To visualize complex formation between vWF and the aptamer, the latter was conjugated with the Cy5 fluorescent label. Cultured human umbilical vein endothelial cells (HUVEC) were stained with the ARC1779-Cy5 conjugate and imaged with a fluorescence microscope. The images were analyzed with the CellProfiler software. vWF released from the Weibel–Palade bodies was observed as bright dot-like structures of round and irregular shape, the number of which increased several times after HUVEC exposure to histamine or thrombin. Staining with ARC1779-Cy5 also revealed long filamentous vWF structures on the surface of activated HUVEC. vWF secretion by ECs is activated by the second messengers cAMP and Ca²⁺. There is evidence that hydrogen peroxide also acts as a second messenger in ECs. In addition, exogenous H₂O₂ formed in leukocytes can enter ECs. The aim of our study was to determine the effect of H₂O₂ on the vWF exposure at the surface of HUVEC using the proposed method. It was shown that hydrogen peroxide at concentration 100 µM, which is lower than the cytotoxicity threshold of H₂O₂ for cultured HUVEC, increased several times the number of dot-like structures and total amount of vWF exposed on plasma membrane of HUVEC, which suggest that H₂O₂ acts as a mediator that activates exocytosis of Weibel–Palade bodies and vWF secretion in the vascular endothelium during inflammation and upon elevated generation of endogenous reactive oxygen species in ECs.

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KEY WORDS: von Willebrand factor, aptamers, hydrogen peroxide, endothelial cells, secretion

DOI: 10.1134/S0006297921020012

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