Abstract
Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the "protein corona" of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery.
Highlights
Biomarkers are sought as measurable indicators of pathological processes and for treatment monitoring
The significant increase of particle counts was seen with serum-derived Extracellular vesicles (EVs) that differed from acid citrate dextrose (ACD), citrate and EDTA plasma–derived EVs in size classes 0–100 nm (p = 0.002 (ACD), p = 0.001, p = 0.023 (EDTA) and 101–200 nm p = 0.044 (ACD) (S1C Fig)
Because their study disregarded a large bulk of EVs, we readdressed this issue with contemporary analytical EV methodologies
Summary
Biomarkers are sought as measurable indicators of pathological processes and for treatment monitoring. The majority of EV biomarker studies have concentrated on the discovery of cancer biomarkers [2], closely followed by biomarkers for cardiovascular diseases [3]. The seminal study of George et al demonstrated that the concentration of “platelet microparticles”, isolated by centrifugation at 35 000 x g, were higher in serum than in plasma anticoagulated by acid citrate dextrose (ACD), which was attributed to platelet activation during clot formation [7]. Nanoparticles (NPs) are developed for drug delivery solutions (DDS). Due to their fast elimination from the body, synthetic NP and their protein corona have recently been under intensive investigation [9,10,11]. In the present study we compared our proteomics results to NP protein corona studies
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