FIGURE 4.

Human bone marrow MSC (hBMSC)‐derived apoVs activate human platelet functions in vitro. (a) KEGG pathway enrichment analysis of significantly upregulated proteins in apoVs compared to exosomes. The enriched KEGG pathways are presented as a bar chart. The Y‐axis represents KEGG pathways and the X‐axes represent the number of significantly upregulated proteins (top) and enrichment significance (bottom), respectively. hBMSCs, human bone marrow MSCs. hASCs, human adipose MSCs. mBMSCs, mouse bone marrow MSCs. (b) Representative confocal microscopy images showing binding of apoVs (red) to the surface of platelets (green). After co‐culture with PKH26‐labeled apoVs at 37°C for 30 min, platelets were stained with Alexa Fluor 488‐conjugated WGA. Scale bar, 1 μm. (c) Flow cytometric analysis and the corresponding quantification of apoV binding to the surface of platelets. After incubation with PKH26‐labeled apoVs at 37°C for 30 min, platelets were stained with CD41 and CD62P. N = 5 per group. (d) Aggregation analysis showing the aggregation of platelets when treated with apoVs or exosomes. The maximal aggregation ratio (MAR) was calculated. PBS was used as the negative control, whereas EPI was used as the positive control. Notably, MAR values lower than the detection range of the analyzer were recorded as “0”. EPI, epinephrine. N = 3–4 per group. (e, f) Representative scanning electron microscopy (SEM) images (e) and confocal microscopy images (f) showing the morphological change of platelets after incubating with apoVs or exosomes. For confocal microscopy, platelets were stained with WGA (green). PBS was used as the negative control, whereas ADP was used as the positive control. ADP, Adenosine diphosphate. Scale bar, 1 μm. (g) Flow cytometric analysis and the corresponding quantification of the percentages of CD41⁺ and CD62P⁺ platelets. The platelets were incubated with apoVs or exosomes at 37°C for 30 min, and stained with CD41 and CD62P. N = 3 per group. (h) Representative platelet spreading and the corresponding quantification of fold changes relative to the negative control group. After incubation with apoVs or exosomes at 37°C for 30 min, platelets were placed on fibrinogen‐coated glass coverslips at 37°C for 90 min followed by staining with WGA (green). Scale bar, 10 μm. N = 3 per group. apoVs, apoVs derived from hBMSCs; Exos, exosomes derived from hBMSCs. Data are presented as mean ± standard deviation (SD). Statistical analyses were performed by Student's t‐test (two‐tailed) for two group comparisons and one‐way ANOVA with Tukey's post hoc test for multiple group comparisons. NS, not significant; **p < 0.01; ***p < 0.001