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.

Hyperfibrinolysis increases blood–brain barrier permeability by a plasmin- and bradykinin-dependent mechanism

“…Cleavage of endogenous HMWK was measured in blood samples from a cohort (n 5 8) of tPA-injected patients ( Figure 7A). 25 First, we analyzed the cleavage of HMWK in those patients at different points before (0 hours) and after (1, 2, 12, and 24 hours; Figure 7B) tPA infusion. We observed that tPA infusion rapidly triggers HMWK cleavage ( Figure 7C-E).…”

Analysis of the thrombotic and fibrinolytic activities of tumor cell–derived extracellular vesicles

“…[36][37][38] Because we have observed that exosomes and MVs from some tumor cells expressed plasminogen receptors and activators (supplemental Figure 2), it was reasonable to suspect that these vesicles might also display fibrinolytic activity. To measure the fibrinolytic activity in vitro, we measured the ability of the exosomes and MVs to digest a pure fibrin clot that was formed by incubating fibrinogen with thrombin ( Figure 4; supplemental Figure 3) or a plasma clot that was formed by incubating human platelet-poor plasma with thrombin ( Figure 5; supplemental Figure 4).…”

Coupling Analytical Methods for Detection of Microparticles: The Possibilities for Improvement

“…Nowadays, there are several methods that currently used for the detection of number (flow cytometry technique, optical microscopy, Nanoparticle Tracking Analysis (NTA), dynamic light scattering) and characterization of structure and features (electronic and atomic force microscopy, fluorescent microscopy, Surface Plasmon Resonance (SPR) technique) of MPs [15,16].…”

“…Flow cytometry technique with polystyrene beads is gold standard to determine the MP sizes that has now standardized by the Scientific Standardization Committee collaborative workshop of the International Society of Thrombosis and Hemostasis [16]. However, this method of size assessment based on SSC has a low resolution of MPs that is roughly estimated to be between 60 and 200 nm, dependent on the vesicle size [17].…”