233. Residual Platelets in stored serum and plasma samples Release Small Extracellular Vesicles Ex-vivo
Creators: name: Malgorzata Malys1
name: Köller1, Maximilian
name: Christof Aigner1, 2
name: Schachner1, Helga
name: Rees1, Andrew
name: Kain1, Renate
Publisher: name: Zenodo
Year: 2022
Subjects: Vasculitis
ANCA
Abstracts
Giant Cell Arthritis
MPA
GPA
EGPA
Takayasu
IgA vasculitis

description: <strong>Background:</strong> Small extracellular vesicles (sEV) are membrane bound particles released from cells that act like messengers between them. They are found in biological liquid including blood, and are particularly abundant in systemic disease such as ANCA-associated vasculitis (AAV). SEVs are composed of protein, lipid and RNA cargo and play a crucial role in cell-cell communication. Their composition varies with cell activation or injury, which potentially could be used as a novel and powerful tool to monitor disease activity and predicting relapse in AAV. Isolating pure sEV from human blood is a challenge due to co-purification of plasma proteins and lipoproteins, but we have optimised protocol for doing this (Malys et al). Here we used this method to enable us to characterise proteins expressed on the surface of sEV in human blood samples with the objective of identifying their cellular origin. <strong>Methods:</strong> Serum, plasma, and platelet poor plasma (PPP) samples were prepared from blood taken from individual donors. These were then used to purify sEV by DUC, either immediately or after storage for 14 days at either 4°C or - 80°C. Concentration and size were Any measured by nanoparticle tracking analysis (NTA) while vesicle morphology, size and CD9 positivity were evaluated by transmission electron microscopy (TEM). SEV were further analysed by western blotting and MACSPlex – bead-based antigen binding array – in order to profile their protein expression. <strong>Results:</strong> There was no difference in the number or diameter of sEV isolated from fresh serum, plasma, and PPP, or from samples stored at -80°C. By contrast, MACSPlex analysis showed that molecules associated with platelets (including CD9, CD63, CD41b, CD42a and P-selectin) were more abundant in sEV preparations purified from fresh serum than fresh plasma, platelet marker abundance also increased in sEV from stored plasma; these changes were greatly attenuated in sEV from PPP. Immuno-EM images from serum and stored plasma samples showed increased CD9 expression. Together, these data suggest that a substantial proportion of sEV detected in serum and stored plasma samples are generated ex-vivo platelets. We confirmed this in experiments in which we deliberately activated platelets by addition of Ca++. TEM from these experiments also identified vesicles with diameters &lt;70nm similar in size to alpha granules released from activated platelets, and below the detection limit of NTA analysis. <strong>Conclusions</strong>: We have shown that serum and stored plasma contain similar populations of sEV, substantial portion of which were not present in the circulation but were released from platelets ex-vivo; this phenomenon can be attenuated by purifying sEV from fresh plasma or from PPP. Our findings have obvious implications for the use of sEV as biomarkers. <strong>Disclosures:</strong> The authors declare no conflict of interest. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 813545.