Assessing Extracellular Vesicle Turnover In vivo Using Highly Sensitive Phosphatidylserine-Binding Reagents

Abstract

Phosphatidylserine (PS) is a well-established marker for apoptotic cells and activated platelets, and it is also found on extracellular vesicles (EVs). However, the frequency of PS+ EVs remains a topic of debate, probably due to the distinct lipid composition of EVs, which varies with the source material. This variation underscores the need for highly sensitive detection reagents to accurately differentiate between PS+ and PS- EVs. To address this issue, we compared several PS-binding reagents for the staining of blood EVs, including milk fat globule factor E8 (MFG-E8, lactadherin), a derivative of MFG-E8, and Annexin V, the most used compound in the literature. Here, we demonstrate the predominance of PS+ EVs in both murine and human blood and reveal significant differences in the affinities of the PS-binding proteins. With approximately 90% of the detected blood EVs being PS+, we utilized a derivative of MFG-E8 for in vivo monitoring of both circulating and cell-bound EVs, providing a novel tool to analyze endogenous EV kinetics. Our findings showed that murine PS+ plasma EVs were rapidly cleared from circulation ([~]50% after 30 minutes), yet they remained present on splenic B cells and monocytes/macrophages. In conclusion, MFG-E8-based reagents enable externalized PS to serve as a universal EV marker, greatly improving EV isolation, standardizing EV quantification, and facilitating diagnostic EV monitoring and disease biomarker discovery.