Generation and characterization of platelet-derived extracellular vesicle analogues from lyophilized platelets

Platelet-derived extracellular vesicles (P-EV) are thought to facilitate the transfer of information from platelets to target cells, playing a role in both physiologic and pathophysiologic processes, particularly in regulating immune responses and healing processes. In addition, P-EV show promise as drug carriers and biomarkers for disease. However, the procedures for isolation, purification and fluorescent labeling of P-EV remain unstandardized. Moreover, the requirement to use freshly obtained platelets for generating EV presents a logistical challenge for their study. In this study, we isolated, characterized, and compared P-EV analogues by sonication of freshly obtained and lyophilized platelets, investigated fluorescent labeling methods, and monitored cellular uptake. We found that P-EV analogues derived from fresh or lyophilized platelets showed similar characteristics regarding size, surface proteins and content. Among the fluorescent labeling methods, CFSE and DiO-C6 were most effective in labeling P-EV analogues from both fresh and lyophilized platelets. All labeling methods led to an increase in P-EV analogue's size, with CFSE and DiO-C6 resulting in the smallest increase. The addition of P-EV analogues to cultured immortal endothelial cells revealed that P-EV analogues were effectively internalized and directed to the lysosomal compartment. The results indicate that P-EV analogues from lyophilized platelets have similar functional properties as those from freshly isolated platelets and these are retained after labeling with CFSE. Thus, lyophilized platelets can serve as a source of P-EV analogues for functional studies.