title: Nano-zymography Using Laser-Scanning Confocal Microscopy Unmasks Proteolytic Activity of Cell-Derived Microparticles.
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.
authors.affiliation: 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.
authors.forename: Aurélien
authors.initials: A
authors.lastname: Briens
authors.affiliation: 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.
authors.forename: Maxime
authors.initials: M
authors.lastname: Gauberti
authors.affiliation: 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.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Parcq
authors.affiliation: 2. Neurovascular Research Laboratory, Vall d'Hebron Research Institute, Barcelona, Spain.
authors.forename: Joan
authors.initials: J
authors.lastname: Montaner
authors.affiliation: 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.
authors.forename: Denis
authors.initials: D
authors.lastname: Vivien
authors.affiliation: 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.
authors.forename: Sara
authors.initials: S
authors.lastname: Martinez de Lizarrondo
mesh_terms: D000818:Animals; D055252:Cell-Derived Microparticles / Q000737:chemistry* / Q000201:enzymology*; D004195:Disease Models, Animal; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods*; D010447:Peptide Hydrolases / Q000032:analysis; D010949:Plasma / Q000737:chemistry; D020521:Stroke / Q000473:pathology
chemical_list: D010447:Peptide Hydrolases
keywords: Extracellular vesicles; MPIO; Optical Imaging; Phosphatidylserine; exosomes; fibrinolysis; inflammation