The Proteomic Characterization Of Platelets And Platelet-derived Exosomes Under Different Activation Pathways

BackgroundPlatelet-derived exosomes(PEXOs)are heterogeneous and bilayer membrane molecules secreted by platelets,which not only serve as important mediators of intercellular communication and cargo transport,but also can regulate many biological processes such as hemostasis,tissue repair and immune response.PEXOs have nanoscale size,low immunogenicity and local release capacity,can cross the tissue barrier,achieve a wider range of regulation.Due to its stable preservation and transport,PEXOs have become ideal substitute for platelet-rich plasma(PRP),and promising drug carrier.The characterization of PEXOs are affected by many factors,especially the activation conditions of platelets.Previous studies did not pay attention to physical activation methods,while chemically activating methods increases costs to a certain extent.The clinical use of PEXOs is still limited due to its main drawbacks that are the lack of standard preparation methods,the variability among separation and activation methods.Exosomes can differentially package cargos from the mother cell during formation and secretion,but differences between PEXOs and platelets have not been fully investigated.Therefore,this study aim to analyze the proteomic characterization of PEXOs induced by different activation conditions,including repeated freezing-thawing.We aim to investigate the differences in proteins between PEXOs and platelets and to explore the potential taregets of PEXOs for therapeutic intervention in tissue repair and anticoagulation.MethodsPlatelets were isolated by differential centrifugation from freshly drawn,citrate anticoagulation blood obtained from healthy volunteers.Platelet suspensions were activated using five different activation conditions.The ctrl group added no extra stimulus,and the other four groups were treated with adenosine diphosphate(ADP),thrombin,Ca2+ionophore and freeze-thaw cycles(FT)to activate platelets.PEXOs were isolated by differential centrifugations,and then were determined by nano-flow cytometry and electron microscopy.The protein markers of PEXOs also were identified by western-blot.The protein concentration and content of PEXOs were also detected.The proteomic characterization of PEXOs and the platelet suspensions were analyzed by data-independent acquisition mass spectrometry(DIA-MS)and then validated by parallel reaction monitoring(PRM)mass spectrometry.Results1.Identification of PEXOsFlow Nano Analyzer showed that the main size peak of all groups of PEXOs is between 75-85nm,85%-95%of PEXOs were<100nm.PEXOs were cup-holder-like bilayer membrane vesicles under transmission electron microscope.WB showed that CD9,CD81,TSG101 and CD41 proteins were positive,while calnexin is negative.These results suggested that these vesicles were PEXOs.2.The biological characterization of PEXOs under different activation pathwaysThe concentration of PEXOs in the FT group was the highest,higher than that in the control group and other groups(P<0.05).The protein concentration of PEXOs in the FT group(1.11±0.51)gg/μL was higher than that in the control group(0.32±0.39)μg/μL,ADP group(0.41±0.31)μg/μL and thrombin group(0.38±0.37)μg/μL,the differences are statistically significant(p<0.05).The protein concentration of FT group was higer than the Ca2+ionophore group(0.51±0.41)μg/μL,(p>0.05)and platelets(0.79±0.52)μg/μL,(p>0.05),The total protein of FT group(125.40±58.32)μg was higher than that in other groups(the Ctrl group 25.53±25.96 vs ADP group 37.24± 15.73 vs thrombin group 36.28±24.18 vs Ca2+ionophore group 47.09±23.29 vs platelets 77.78±32.95)μg,the differences are statistically significant(p<0.05).The activation pathway regulates the quality of PEXOs.Regarding the concentration or protein concentration or protein content of PEXOs,the order in our study is FT group>Ca2+ionophore group>ADP group≈thrombin group>ctrl group.3.DIA-MS analysisMS detected 3216 proteins,1090 proteins were expressed in all groups,and these proteins were mainly involved in signal transduction,immune system and cancer-related signaling pathway.26 proteins were expressed in all groups of PEXOs but not in platelets.Each group of PEXOs expressed unique proteins which had not expressesd in other groups.Compared with platelets,the up-regulated proteins in the adenosine diphosphate group,thrombin group and calcium ionophore group were significantly enriched in the complement and coagulation cascade.PDGFB,PDGFD and a group of proteins involved in extracellular matrix interacting increased in the thrombin group.4.PRM validation study 30 proteins were selected based on the analysis of DIA-MS and then were validated by PRM study.The thrombin group and FT group expressed the same 15 target proteins,while the ADP group expressed 14 target proteins except EGF,platelets expressed 16 target proteins.The platelets and PEXOs have 12 common proteins,such as A2M,PDGFB,PDGFD,FN1,SPARC,THBS1 and PF4.EVA1B was expressed in all three PEXOs groups but not in platelets.We tried to detect the unique proteins of ADP group(PLA2G2A),thrombin group(RPS15)and FT group PEXOs(EXOC5 and COMMD3)based on DIA-MS analysis,failed in PRM,TMED10 decreased in thrombin group,9 proteins(THBS1,PF4,SPARC,PDGFB,PDGFD,A2M,SERPINA1,SERPINC1,APOA1)increasd,compared with platelets.Except SERPINA1,the regulation type were all consistent with that in DIA-MS.ConclusionThe protein differentially packaged process of PEXOs is closely related to the activation pathways of platelets,which can not only regulate their proteomic characterization,but also determine their function.The greater stimulus intensity,the more protein contents and types.There are non-mother-cell derived proteins in PEXOs,which may be related to the formation process and roles of exosomes.E ach group of PEXOs has unique proteins which only expressd in one group.Freeze-thaw cycles can induce a large amount protein-rich PEXOs which has little difference in protein profiling compared with platelets,it may be an ideal condition for the use of study and preparation of PEXOs.Compared with platelets,PDGFB and PFGFD increased in PEXOs induced by thrombin,and they may have therapeutic effects in tissue repair through interaction of extracellular matrix.