Comparison and Investigation of Exosomes Derived from Platelet-Rich Plasma Activated by Different Agonists

Abstract

PRP-Exos are nanoscale cup-shaped vesicles that carry a variety of proteins, mRNAs, microRNAs, and other bioactive substances. PRP-Exos can be formed through several induction pathways, which determine their molecular profiles and facilitate their tailormade participation in intercellular communication. Currently, little is known on how the PRP-Exos activation method influences the quality and quantity of PRP-Exos. The present study aims to observe and analyze the number, profile, and growth factors of PRP-Exos through TEM, Nanoflow, and WB after PRP activation and compare the difference in function of PRP-Exos on HUVECs, with different stimuli (calcium gluconate, thrombin, or both). We found that PRP activated with both thrombin and calcium gluconate harvested the highest concentration of exosomes [(7.16 ± 0.46) × 1010 particles/ml], compared to thrombin group [(4.87 ± 0.15) × 1010 particles/ml], calcium gluconate group [(5.85 ± 0.43) × 1010 particles/ml], or saline group [(7.52 ± 0.19) × 109 particles/ml], respectively ( P < 0.05) via Nanoflow analysis. The WB analysis showed that cytokines (VEGF, PDGFBB, bFGF, TGF-β) are differentially encapsulated in PRP-Exos, depending on the PRP stimulus, in which the mixture-PRP-Exos yielded the highest concentration of cytokines. In the function assay of PRP-Exos on HUVECs, the mixture-PRP-Exos promoted HUVECs proliferation, increased HUVECs migration, promoted the formation of vessel-like by HUVECs via the AKT ERK signal pathway more dramatically, compared with other groups. In summary, our studies showed that PRP activated by the mixture of calcium gluconate and thrombin harvested the best quality of exosomes which had the top biological functions. This study provides a protocol for selecting appropriate PRP activators to obtain high-quality exosomes for future applications.

Isolation of Platelet-Derived Exosomes from Human Platelet-Rich Plasma: Biochemical and Morphological Characterization

“…However, Coomassie-staining revealed clear differences in the band profiles between the calcium-activation versus baseline conditions, confirming that calcium impacts on the composition of the cargo release profile of PLT-Exos. To compare the presence of non-exosome and exosome-specific proteins in the different fractions, we chose a set of markers according to widely accepted criteria [ 11 , 12 , 13 , 14 , 25 , 32 , 33 ]. Interestingly, vesicles isolated from calcium-activated platelets were virtually devoid of plasma membrane (caveolin-1), endoplasmic reticulum (calreticulin), mitochondria (Human Porin proteins VDAC1 and 3, cyclophilin F, cytochrome C), lysosome (lamp-1), and early endosome (Rab5) markers, whereas those isolated from unstimulated platelets exhibited clear immunoreactivity for all of these proteins.…”

“…Indeed, human PLT-Exos have been shown to increase cell proliferation and migration of mesenchymal stromal cells (MSCs) from human bone marrow [ 10 ], to prevent apoptosis-caused glucocorticoid-associated endoplasmic reticulum stress in a rat model of osteonecrosis [ 11 ] and to promote re-epithelization of chronic ulcers in a diabetic rat model [ 12 ]. In turn, homologous PRP-Exos have shown to promote proliferation and inhibit apoptosis of rabbit chondrocytes [ 13 ] and to promote the formation of vessel-like structures from cultured human umbilical vein endothelial cells while increasing their proliferation and migration rates [ 14 ].…”

“…In this sense, the methodological variability in the different steps for the isolation of PLT-Exos can influence the outcome concerning the yield, purity, and specific cargo composition [ 20 ]. Indeed, reported methods vary considerably, ranging from the direct purification from activated PRP, under the assumption that most of the exosomes present in PRP are of platelet origin [ 13 , 14 ], to isolating them from lysates of non-activated platelets [ 10 ] or from supernatants of activated platelets that have previously been separated from PRP [ 11 , 12 , 25 ].…”

Characterization and Therapeutic Use of Extracellular Vesicles Derived from Platelets

“…The dynamic content of PL-EVs including proteins from the platelet membrane, cytosol, organelles, adhesion receptors, coagulation factors, transcription factors, growth factors, active enzymes, cytokines, chemokines and their receptors is mainly dependent on the mechanism of platelet activation, the agonist used and the time of stimulation. Previous knowledge includes the formation of PL-EVs during the process of platelet activation by soluble agonists, activators of second messengers (such as calcium ionophores and phorbol esters), physiological agonists (pathogens, high shear stress, contact with surfaces, low temperature and storage) or during platelet senescence and apoptosis, thrombus degradation and during megakaryocytosis ( Figure 2 ) [ 95 , 96 ]. Soluble agonists include collagen, von Willebrand factor, adenosine diphosphate (ADP), thrombin, fibrinogen, fibronectin, serotonin and platelet-activating factor [ 97 , 98 ].…”

“…Different processes of platelet activation may lead to the formation of heterogeneous PL-EV populations with different surface marker expression and protein profiles, which may affect their role in intercellular communication [ 99 ]. Several comparative studies have been performed, which compared the effect of different activators on platelet activation and the subsequent release of exosomes [ 49 , 95 , 100 , 101 ]. For example, EVs from platelets activated in vitro with ADP contain different protein cargo in comparison with those activated by collagen or collagen and thrombin [ 99 ], implying that these EVs could also have different functions.…”