Assessing Extracellular Vesicle Turnover In Vivo Using Highly Sensitive Phosphatidylserine‐Binding Reagents

Extracellular vesicles (EVs) are emerging as crucial players in cell communication and hold great promise as biomarkers and therapeutic tools. However, their diversity makes it challenging to detect, classify, and utilize them effectively, which limits their clinical applicability. A key challenge is the lack of reliable markers to identify EVs consistently. In this study, a novel high-affinity phosphatidylserine (PS)-binding reagent is introduced for EV analysis. PS is known as a marker of apoptotic cells and activated platelets, but its presence on EVs is debated due to variations in lipid composition. By comparing multiple PS-binding reagents, including MFG-E8 derivatives and Annexin V, it is demonstrated that ≈90% of EVs in human and mouse blood carry PS. Using the optimized reagent, the first in vivo insights into EV turnover are provided, showing that PS+ EVs in mouse blood are rapidly cleared (≈50% within 30 min) but persist on immune cells in the spleen. This discovery increases the potential of EVs as disease biomarkers and therapeutic targets by improving EV detection and isolation as well as opening the door for standardized quantification and diagnostic monitoring.