Fig 1.
Serum sEV fractions contain more EV particles than plasma sEV fractions in mouse blood.
(A) Schematic representation of the preparation methods of plasma and serum. Plasma was separated from anticoagulant-treated blood by removing hemocytes and other precipitates by centrifugation. Serum was separated from coagulated blood by removing blood clots by centrifugation. (B) Transmission electron micrographs of particles in the sEV fractions derived from plasma and serum. Scale bars, 100 nm. (C-E) NTA of the sEV fractions derived from plasma and serum. A plot showing the size distribution of the EV particles (C) and bar graphs showing the numbers (D) and median diameters (E) of the particles in the sEV fractions isolated from plasma and serum. Data are shown as the average ± SEM (plasma, n = 5; serum, n = 6). **P < 0.01, Student t-test. n.s., not significant. Experiments were performed independently at least three times, and representative data are shown. In C and D, particle numbers are normalized to original blood volumes.
Fig 2.
Anticoagulants have no detectable effects on numbers and diameters of EVs in plasma.
(A-B) Bar graphs showing numbers (A) and median diameters (B) of the EVs that were isolated from plasma prepared using EDTA, citrate, and heparin. Data are shown as the average ± SEM (n = 5). Statistical analysis was performed by one-way ANOVA followed by the Tukey multiple comparisons test. n.s., not significant.
Fig 3.
Proteomic analysis demonstrates substantial enrichment of platelet-associated proteins in serum EVs.
(A) Venn diagram of the proteins identified in the sEV fractions derived from plasma and serum (n = 6) by LC-MS/MS analysis. (B) GO cellular component of the proteins identified in plasma EVs and serum EVs. P-values from the modified Fisher’s exact test are shown as bar graphs. (C) Principal component analysis of the dataset of proteins identified in plasma EVs and serum EVs. (D) GO biological process of the proteins that are upregulated or exclusively detected in serum EVs. (E) Heat map analysis of proteins with platelet-associated GO terms that were identified in our LC-MS/MS analysis.
Table 1.
Platelet-associated proteins detected in serum EVs.
Fig 4.
Platelet-specific proteins are abundantly detected in sEV fractions derived from serum of mouse blood.
(A-D) Western blot analysis of the sEV fractions derived from plasma and serum using antibodies against EV marker proteins and platelet-specific proteins (A) and their densitometric analyses (B-D). Equal amounts of proteins were loaded onto each lane and analyzed (n = 3). The level of CD63 (B) and the levels of EV markers CD9 and Hsc70 relative to CD63 (C) showed no statistical difference between plasma and serum EVs. In contrast, platelet-specific GPIIb, GPIIIa, and PF4 were significantly increased in the serum sEV fractions (D). The asterisk in (A) indicates a nonspecific signal. (E) Optiprep density-gradient centrifugation analysis of the serum sEV fraction with EV marker proteins and platelet-specific proteins. GPIIb and GPIIIa were both detected in the fractions at the same density as the sEV, and cofractionated with EV markers. Data are shown as the average ± SEM. *P < 0.05, **P < 0.01, Student t-test. n.s., not significant.
Fig 5.
Serum EVs contain high levels of platelet proteins in human blood.
(A) Western blot analysis of the sEV fractions derived from plasma and serum of 5 individuals, using antibodies against EV markers and platelet-specific proteins. Equal amounts of proteins were loaded onto each lane and analyzed. (B) Bar graph showing relative protein levels of CD63 that were calculated from the band intensity of the Western blot image in (A). (C-D) Bar graphs showing the levels of the EV marker CD9 (C), and platelet-specific markers GPIIb, GPIIIa and PF4 (D), relative to that of CD63. Whereas the level of CD9 was comparable between the sEV fractions from plasma and serum, all the platelet markers showed a clear tendency of increased levels in serum EVs. (E) NTA results showing the numbers (left) and median diameters (right) of the particles in the sEV fractions derived from human plasma and serum. (F) Comparison of the sEV fractions derived from mouse and human blood by Western blot analysis using antibodies against EV markers and lipoprotein ApoA1. The sEV fractions isolated from human blood contain very low levels of the EV markers CD63 and Hsc70, but a high level of ApoA1, implying a large difference in particle population between human and mouse blood. Data are shown as the average ± SEM. Student t-test. n.s., not significant.
Fig 6.
Platelet-associated proteins are generally enriched in serum EVs.
(A-D) Bar graphs showing modified Fisher’s exact P-values of the GO biological process terms associated with platelets (A), complement activation (B), endocytosis/phagocytosis (C), and immune response (D), which were calculated from the proteomic data of plasma EVs (P1, Zheng et al.; P2, Yentrapalli et al.; P3, Bezdan et al.), and serum EVs (S1, Zhong et al.; S2, An et al.; S3, Ding et al.; S4, Luo et al.) reported from other groups. The GO terms associated with platelets were significantly enriched in serum EVs compared with plasma EVs, whereas the other GO terms showed almost comparable enrichment among the studies analyzed.