TABLE 3.
Functional assays for EVs.
| General aspects | Reviewed in (Coumans et al., 2017; Théry et al., 2018) | ||
|---|---|---|---|
| Biological function | Methods | Pitfalls/Limitations | References |
| Coagulatory functions | |||
| Clotting time | • Measuring clotting time of plasma upon FX activation to determine EV-PS activity | • Restricted to plasma samples | (Exner et al., 2003; Luddington and Baglin, 2004) |
| Thrombin generation | • Measuring thrombin generation after capture of EV-PS on annexin-V coated ELISA plates upon addition of TF and phospholipids | • Requires an inhibitor of contact activation, e.g., CTI | (Hemker et al., 2002; Jy et al., 2004) |
| • Measuring coagulant EV-TF in terms of thrombin, fibrin of FXa generation | • Use of specific antibodies to block TF coagulant activity | (Hellum et al., 2012; Thaler et al., 2012; Thaler et al., 2013; Tatsumi et al., 2014) | |
| • TF-dependent FXa generation upon addition of FVII and phospholipids | • PS-quantification only when PS source is restricted to EVs | Summarized in (Agouti et al., 2015; Hisada et al., 2016; Hisada and Mackman, 2019) | |
| • Fibrin generation in plasma determines EV’s PS and TF activity | • Requires concentration of plasma EVs by centrifugation, but concentration and isolation of EVs contributes to poor reproducibility of the respective functional test | Berckmans et al. (2011) | |
| Fibrinolysis | |||
| Plasmin generation | • Measurement of plasmin generation using plasmin-selective chromogenic or fluorogenic substrates | • No standards available | (Lacroix et al., 2007; Miszta et al., 2020) |
| • Needs controls specific for plasmin generation, such as α2-antiplasmin or an inhibitory antibody against urokinase | |||
| Cellular functions (including EC-functions) | |||
| Migration | • Effects of EVs on trans-well migration and wound healing models (e.g., scratch assays) | • Cell culture or organoid models might not reflect the in vivo situation sufficiently | (Silva et al., 2017; Kriebel et al., 2018) |
| Proliferation | • Effects of EVs on cell numbers | Zhong et al. (2020) | |
| • DNA-synthesis (BrdU incorporation etc.) | |||
| Formation of spheroids and sprouts | • Sprouting of ECs from beads | Hood et al. (2009) | |
| • Formation of 3D-organoids (e.g., of cancers cells) | |||
| TEER (Transendothelial Electrical Resistance) | • TEER measurement using HUVECs or isolated primary endothelial cells | Dean et al. (2009) | |
| Tube formation | Skog et al. (2008) | ||