Theranostics 2016; 6(5):610-626. doi:10.7150/thno.13757

Research Paper

Nano-zymography Using Laser-Scanning Confocal Microscopy Unmasks Proteolytic Activity of Cell-Derived Microparticles

Aurélien Briens1*, Maxime Gauberti1*, Jérôme Parcq1, Joan Montaner2, Denis Vivien1, Sara Martinez de Lizarrondo1✉

1. Institut National de la Santé et de la Recherche Médicale (INSERM), INSERM UMR-S U919, Serine Proteases and Pathophysiology of the Neurovascular Unit, GIP Cyceron, Université Caen-Normandie, Caen, France.
2. Neurovascular Research Laboratory, Vall d'Hebron Research Institute, Barcelona, Spain.
*A.B. and M.G participated equally to this work.

Abstract

Cell-derived microparticles (MPs) are nano-sized vesicles released by activated cells in the extracellular milieu. They act as vectors of biological activity by carrying membrane-anchored and cytoplasmic constituents of the parental cells. Although detection and characterization of cell-derived MPs may be of high diagnostic and prognostic values in a number of human diseases, reliable measurement of their size, number and biological activity still remains challenging using currently available methods. In the present study, we developed a protocol to directly image and functionally characterize MPs using high-resolution laser-scanning confocal microscopy. Once trapped on annexin-V coated micro-wells, we developed several assays using fluorescent reporters to measure their size, detect membrane antigens and evaluate proteolytic activity (nano-zymography). In particular, we demonstrated the applicability and specificity of this method to detect antigens and proteolytic activities of tissue-type plasminogen activator (tPA), urokinase and plasmin at the surface of engineered MPs from transfected cell-lines. Furthermore, we were able to identify a subset of tPA-bearing fibrinolytic MPs using plasma samples from a cohort of ischemic stroke patients who received thrombolytic therapy and in an experimental model of thrombin-induced ischemic stroke in mice. Overall, this method is promising for functional characterization of cell-derived MPs.

Keywords: Extracellular vesicles, Phosphatidylserine, Optical Imaging, fibrinolysis, MPIO, exosomes, inflammation

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How to cite this article:
Briens A, Gauberti M, Parcq J, Montaner J, Vivien D, Martinez de Lizarrondo S. Nano-zymography Using Laser-Scanning Confocal Microscopy Unmasks Proteolytic Activity of Cell-Derived Microparticles. Theranostics 2016; 6(5):610-626. doi:10.7150/thno.13757. Available from http://www.thno.org/v06p0610.htm