| BankgroundPlatelet transfusion is an important clinical method for the treatment of low platelet count.At present,platelet transfusion completely depends on volunteer donation.Transfusion platelets are usually collected by the blood station and kept properly to maintain the complete physiological activity.At present,the mechanism of platelet storage lesion is still unclear,and there are still some defects in the process of platelet preservation,such as short preservation period,easy to be polluted,decreased vitality and so on.These factors lead to the huge waste of platelet transfusion and seasonal fluctuations of platelet storage.At present,platelet preservation conditions mainly include:room temperature oscillation;low temperature(4℃);deep hypothermia(-80℃);freeze drying and so on.The room temperature oscillation is a common way of platelet preservation.However,at room temperature,platelets are prone to bacterial pollution and lactate production caused by metabolic disorders,and the preservation time is usually less than 5 days;Deep hypothermia can save platelets for a long time,but the platelet activity is affected by cryopreservation agents,which makes platelets activate and lose their clinical application value.The process of rehydration of frozen dried platelets can change the morphology and surface glycoprotein of platelets,but can not avoid reducing the function.Relatively speaking,platelets stored at low temperature(4℃)can maintain high coagulation activity for a long time,cause lactic acid aggregation,pH reduction and other problems to a minimum,and significantly reduce the occurrence of bacterial contamination.At the same time,it is convenient for blood bank operation and saves operating expenses.However,cryopreserved platelets can be rapidly combined with and removed from the liver cells(AMR,Ashwell Morell receptor)and Kupffer cells(integrin αMβ2),which limits its application in preventive infusion.Platelet storage lesion(PSL)refers to a series of morphological,functional and metabolic changes of platelets during storage due to lack of energy and accumulation of metabolites.In view of the advantages and disadvantages of the above platelet storage methods,it is of great significance to explore the optimal storage conditions of platelets and reduce the damage of platelet storage.Autophagy is a widespread degradation/recycling system in eukaryotes.Cells degrade intracellular proteins and organelles in the absence of energy and amino acids,and use the degradation products to synthesize new proteins or organelles.As the smallest akaryote cell in circulation,platelets are also capable of autophagy.Although autophagy has been shown to be related to platelet generation,longevity and activation,the role of autophagy on platelets remains unclear.In the process of platelet storage in vitro,due to the lack of energy,platelet autophagy will occur,but whether autophagy will affect platelet storage lesion and its mechanism is still unclear,and whether it can reduce platelet storage lesion by interfering with autophagy is also unclear.objectiveIn this study,we will:1.To explore the changes of autophagy state during platelet storage;2.To explore the occurrence of platelet storage lesion during platelet storage;3.To explore the effect of autophagy intervention on maintaining normal physiological activity of preserved platelets and the method of improving platelet preservation cycle through autophagy intervention.We hope to further optimize the platelet preservation conditions and prolong the platelet preservation cycle through this study.Method1.Separation of platelet rich plasma(PRP)Fresh blood was collected and platelet rich plasma(PRP)was separated and stored at different time points2.Western BlotDifferent PRPs were collected and placed for different times,and the expression levels of autophagy related proteins LC3 Ⅱ、LC3Ⅰ、LC3 Ⅱ/LC3Ⅰ and P62 were detected by Western Blot to investigate the autophagy changes of platelets placed for different times.The PRPs treated with different treatments were collected and placed at different times,and the expression levels of autophagy related proteins LC3 Ⅱ、LC3 Ⅰ、LC3 Ⅱ/LC3Ⅰ and P62 were detected by Western Blot to explore the influence of autophagy intervention reagents on the occurrence of autophagy state during plate storage3.Flow cytometryPRPs treated with different treatments were collected and stored at different times.The surface receptor abdication(GPVI,CD42b),particle release(CD62p),sialic acid modified abdication(RCA)and phosphatidylserine exposure(Annexin V)during platelet storage were detected by flow cytometry.The effects of autophagy intervention reagents Rap and Baf on surface receptor abdication,particle release,sialic acid modification abdication and phosphatidylserine exposure during platelet storage were detected by flow cytometry4.Enzyme-linked Immunosorbent Assay(ELISA)Collect PRPs for different processes placed at different times,the neuraminidase activities,ADAM 10 and ADAM 17 activities of PRP treated at different times were detected by ELISA5.Platelet aggregation testPRP treated with autophagy intervention reagent and PRP treated with PBS were collected and placed at different time points.Platelet activity of different treatment groups was detected by platelet aggregator at different time points6.Statistical analysisSPSS 21.0 statistical software and GraphPad Prism were used to analyze the experimental data.The experimental data were expressed as Mean ± SEM.The comparison of quantitative data between the two groups was performed by T test,and P<0.05 was considered statistically significantResults1.During platelet preservation,there is a continuous autophagy flux2.During the preservation process,the expression of platelet surface receptors CD42b and GPVI decreased3.The activities of ADAM 17 and ADAM 10 in plasma increased slowly during the platelets preservation in 37 degree4.Rapamycin increases autophagy activity of platelets in the early stage of platelet preservation,reduces platelet preservation injury and improves platelet reactivityConclusionThe regulation of platelet autophagy by Rapamycin at low concentration can decrease the cleavage of platelet surface receptors CD42b and GPVI,and thus improve platelet viability... |
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