Comparative phenotyping of surface markers and glycans in murine and human platelet-derived extracellular vesicles.

Background: Platelet-derived extracellular vesicles (PEVs, 100-1000 nm in size) are released from activated platelets. They are important carriers of signaling molecules, lipids, and proteins. They interact with immune and tumor cells and, thus, hold great promise as drug delivery tools. They express a plethora of unique surface glycans, and the majority of PEVs in circulation are nonprocoagulant and phosphatidylserine-negative. Despite an increasing number of studies on PEVs, receptor and glycosylation profiles of murine and human PEVs have not been characterized systematically. Objectives: To characterize large PEVs (l-PEVs) generated from human and murine platelets using double stimulation of glycoprotein VI (GPVI) and PAR1/4 pathways, as well as ionophore A23187. Methods: Surface glycoprotein and glycosylation profiles of human and mouse l-PEVs were determined using flow cytometry, western blotting, and lectin array. Results: Interestingly, murine samples showed a significantly altered glycoprotein expression and glycosylation compared with human l-PEVs. Following stimulation, GPVI, CD41/CD61, and CD62P were expressed to a lower extent on murine l-PEVs. CD42b (GPIb) was not highly abundant on l-PEVs from both species. The pan-inhibition of metalloproteinases resulted in recovery of CD42b but not GPVI on l-PEVs. Moreover, the analysis of surface and the total content of carbohydrates of the l-PEVs showed species-specific surface glycan exposure. We also detected only minor differences in phenotypes of l-PEVs generated from buffy coat or fresh blood and between l-PEVs generated from blood with different types of anticoagulation. Conclusion: We identified specific differences in the phenotype of mouse vs human l-PEVs, which contribute to our understanding on the role of PEVs, which should be considered when designing future PEV studies, for example, in commonly used murine models of disease.