Establishing Phosphatidylserine (PS) as the Universal EV-Marker: Novel highly sensitive PS-specific reagents allow measuring global EV-turnover in vivo

Background

Phosphatidylserine (PS) is a widely established marker for apoptotic cells, but it is also found on extracellular vesicles (EVs). However, it remains controversial as to how frequent PS+EVs are. This might be due to the different PS-contents of EVs depending on the source material. In addition, previously available PS-detection reagents may have only limited sensitivity. To address this problem, we have generated several PS-detection reagents ourselves and compared distinct PS-binding reagents for staining of EVs. Those reagents include MFG-E8, tetramerized MFG-E8 C1 domains (C1 tetramers), and Annexin V.

 

Methods

We isolated EVs from serum and plasma using size exclusion columns and determined the amount of PS+ EVs with different reagents by imaging flow cytometry, bead-assisted flow cytometry and superresolution microscopy. The different reagents showed up to 1000x different binding affinities for PS+ EVs. Using the most sensitive reagent, we determined the in vivo turnover of EVs in the blood by labeling EVs with intravenously administered PS-binding reagent. To determine the kinetics of EV clearance and EV-half-life in vivo, we measured the number of labeled EVs at different time intervals after injection. 

 

Results

We could show that PS+ EVs are highly prevalent in murine and human blood but also noticed striking differences depending on the detection reagent used. The amount of PS+ EVs ranged from 30% (Annexin V) to almost 100% (MFG-E8 and C1-Tetramers), explaining the contradictory reports about PS-positivity of EVs in the literature.

We then used C1-tetramers for in vivo monitoring of circulating EVs and could show that also in vivo labeling revealed that >90% EVs are PS+ in murine plasma and were rapidly cleared from the circulation within 1 hour while still being bound by specific cell types.

 

Conclusion

We demonstrated that surface PS is a universal EV marker present on nearly all (approx. 97%) EVs in blood. Nevertheless, in the analysis of externalized PS on EVs, the selection and titration of PS-detection reagents holds paramount importance, as it has previously resulted in a significant underestimation of PS-positive EVs.

 

Keywords

Phosphatidylserine, MFG-E8, lactadherin, C1 Tetramers, EV-half live

 

Funding/Acknowledgments 

DFG-CRC 1054 (B03, Z01, Z02)

 

Authors

Lavinia Flaskamp1, Tijana Kandic1, Christine Ried1, Jan Kranich1, Thomas Brocker1 (Corresponding Author: Thomas Brocker, brocker[at]lmu[dot]de)

 

1 LMU Munich, Institute for Immunology, Munich, Germany

 

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