Qualitative and Quantitative Comparison of Plasma Exosomes from Neonates and Adults

Abstract

Exosomes are extracellular vesicles that contain nucleic acids, lipids and metabolites, and play a critical role in health and disease as mediators of intercellular communication. The majority of extracellular vesicles in the blood are platelet-derived. Compared to adults, neonatal platelets are hyporeactive and show impaired granule release, associated with defects in Soluble N-ethylmaleimide-sensitive fusion Attachment protein REceptor (SNARE) proteins. Since these proteins participate in biogenesis of exosomes, we investigated the potential differences between newborn and adult plasma-derived exosomes. Plasma-derived exosomes were isolated by ultracentrifugation of umbilical cord blood from full-term neonates or peripheral blood from adults. Exosome characterization included size determination by transmission electron microscopy and quantitative proteomic analysis. Plasma-derived exosomes from neonates were significantly smaller and contained 65% less protein than those from adults. Remarkably, 131 proteins were found to be differentially expressed, 83 overexpressed and 48 underexpressed in neonatal (vs. adult) exosomes. Whereas the upregulated proteins in plasma exosomes from neonates are associated with platelet activation, coagulation and granule secretion, most of the underexpressed proteins are immunoglobulins. This is the first study showing that exosome size and content change with age. Our findings may contribute to elucidating the potential "developmental hemostatic mismatch risk" associated with transfusions containing plasma exosomes from adults.

Keywords: exosomes; neonatal platelets; platelet transfusion; protein S; proteomic; von Willebrand factor.

Figure 1

Workflow diagram. Representation of the sequential steps followed blood extraction to exosomes isolation and characterization, and analysis of results.

Figure 2

Characterization of exosomes. (A) Representative images of plasmatic adult and neonate exosomes from the transmission electron microscopy (TEM) (n = 3/group). The scale bars of images are 200 nm. The acquisition was at 100 kV. The magnification was 100,000×. The images were processed using MIP4 software. (B) Percentage of exosomes in different diameter size ranges (30–50 nm; 50–80 nm; 80–120 nm) in adult and neonate samples. Values represent average population percentage relative to total exosome population ± SD (n = 3/group); (* p < 0.05).

Figure 3

Unsupervised analysis of differentially expressed proteins in plasma exosomes from neonates vs. adults by SWATH MS. (A) Heat map showing hierarchical clustering between adult and neonate exosomes using top 50 differentially expressed proteins. Analysis was carried out in 3 pool samples, each consisting of pooled plasma exosomes from 2 newborns or 2 adults. (B) PCA analysis showing separation of adult (red) and neonate (green) exosome samples. (C) Volcano diagram resulting from comparison of newborn and adult exosomes. Proteins are separated according to the log₂ of the FCh (x-axis) and the −log₁₀ of the p-values based on a two-tailed t-test (y-axis).

Figure 4

Reactome network view of differentially expressed proteins. (A) Hemostasis pathway showing differentially expressed proteins in adults and neonatal exosomes involved in different subpathways (a–g). (B) Vesicle-mediated transport pathway showing differentially expressed proteins in adults and neonatal exosomes involved in membrane trafficking (h) or binding and uptake of ligands by scavenger receptors (i).

Figure 5

Validation of differential protein expression in independent samples. (A) vWF levels detected by ELISA in exosomes of adults (n = 10) and neonates (n = 10). ELISA analysis reveals statistically significant differences in von Willebrand factor (vWF) concentrations (Mann–Whitney U test). **** p < 0.0001. (B) Western blot of protein S in plasmatic exosomes from adults (A; n = 4) and neonates (N; n = 4) (unpaired t-test, one-tailed). Tumor susceptibility gene 101 (TSG-101) was evaluated as exosome-specific marker.