The Platelet Transcriptome And Proteome Is Altered And Prothrombotic Functional Responses Are Increased During Prolonged Hypoxia Exposure At High Altitude

Unique and complex ecological environments and geographical conditions in the plateau cause people who have migrated into these areas suffer from altitude sickness or hypoxia-related diseases.Hypoxia,low pressure,strong solar radiation,large temperature differences between day and night and dryness are the main risk factors of altitude sickness or diseases.Hypoxia extensively and specifically involves many systems of the body,including the cardiovascular,respiratory,nervous,digestive and immune systems,affecting the body’s function,metabolism and morphological structure.Recent studies have identified that hypoxia exposure at high altitudes can also lead to thromboembolic events in migrant populations.Hypoxia exposure at high altitudes can induce thrombosis,which has been confirmed in hypoxia-related activities and diseases,such as high-altitude climbing activities,neonatal hypoxia and patients with obstructive sleep apnea hypoventilation syndrome.However,the causes and molecular mechanisms of hypoxia exposure induced thrombosis are rarely reported.Microarrays are widely used for the analysis of gene expression profiles of various diseases due to its speed,high throughput,high sensitivity and high efficiency.Microarrays were used to examine platelet gene expression profiles in patients with diseases,such as sickle cell disease,systemic lupus erythematosus and primary thrombocytopenia,and it was found that differentially expressed genes contribute to these diseases.In our study,microarray analysis was used to compare differentially expressed genes in platelets of healthy individuals at sea level and high altitude,while the function of the differentially expressed genes were analyzed using bioinformatics methods to understand the possible mechanism of chronic hypoxia exposure induced thrombosis.This study aimed to identify the effect of chronic hypoxia exposure at high altitude on platelet gene expression and function activation,as well as their relationship with thrombosis,which is of high clinical significance and provides further evidence that chronic hypoxia exposure induces thrombosis.Objective: In order to explore the effect of chronic hypoxia exposure at high altitudes on platelet count,transcriptomes,platelet function and their possible molecular mechanisms during thrombosis.This study is in two parts:(1)platelet transcriptome and proteome is alteration and pro-thrombotic functional response increase during prolonged hypoxia exposure at high altitudes.(2)Chronic hypoxia exposure alteration of platelet gene expression and increased activation in rats.Method: 43 apparently healthy males,who had been residing at high-altitudes(HA)continuously for at least two months were enrolled,while 39 healthy males residing at levels up to 50 meters above sea level were enrolled as the control group(SL).After,clinical parameters were identified,whole blood cell and platelet count were measured.Next,using GeneChip? PrimeView? Human Gene Expression Array Affymetrix,differentially expressed genes of platelet mRNA transcriptomes between sea level and high altitude groups were found,and GO and pathway analysis were conducted on the differentially expressed genes.qPCR and western blotting were also performed to validate the differentially expressed genes in platelets.The serum PF4 and ADP concentrations of the two groups were detected using ELISA.Clot retraction,area covered by platelet adhesion to fibrinogen were detected,while transmission electron microscopy was used to observe platelet morphology,intracellular ultrastructure,alpha granule release in both sea level and high altitude groups.Based on the results of part I,the rats were used to further verify the results of tests conducted on humans.A total of 26 male Sprague-Dawley rats were randomly divided into two groups,one group was used as sea level controls(SE,n=13,50 m),while the other group was used as the hypoxia group and were exposed to a simulated altitude of 4000 m(HA,n=13).Real-time fluorescence quantitative PCR and western blotting were used to detect platelet membrane receptor GP4,GP6,GP9,GP1 BB,ITGA2B,ITGB3,P2Y12 and SELP expressions at both mRNA and protein level.The tail bleeding time was compared between the SE and HA group,to evaluate the effect of hypoxia exposure on physiological hemostasis.The PF4 and β-TG expression of the two groups were detected using ELISA.Clot retraction was also compared between the SE and HA groups.Confocal microscopy was used to detect platelet adhesion and spreading on fibrinogen between SE and HA group.Results:(1)Platelet count decreased in the HA group,compared with that of the SL group,which was accompanied by a phenotype change.There were 3,445 genes that met the FDR<0.05 criteria.Compared with the SL group,901 genes were upregulated and 2,544 genes were downregulated in the HA group.GO analysis and biological pathway analysis identified that that the differentially expressed genes were enriched in coagulation,hemostasis,platelet activation,signal transduction,aggregation and degranulation.Based on the bioinformatics analysis,we selected genes that can edit the platelet membrane receptor to be validated using real-time quantitative PCR and western blotting.The expression of GP4,GP6,GP9,ITGA2 B,SELP and PF4V1 were found to have increased at mRNA and protein level in the HA group,compared with that of the SL group(P<0.05).Transmission electron microscopy shows that platelet degranulation is significantly increased and triggers the release of alpha granules in HA healthy individuals,while PF4 and ADP expression were higher in HA group,compared with that of the SL group.Hypoxia exposure increases engagement and adhesion to fibrinogen(P=0.0003),while the clot area was smaller in the HA group than the SL group,regardless whether the duration was 30 min(P=0.0095)or 120 min(P=0.0043).(2)Chronic hypoxia exposure decreases platelet count(P=0.000).The results of the real-time quantitative PCR show that the mRNA expressions of platelets GP4,GP6,GP9,GP1 BB,ITGA2B,ITGB3 and SELP in the HA group of rats were significantly higher than that of SE group rats(P<0.05),but no difference in ADP receptor P2Y12 expression was found between the two groups.The western blotting analysis results show that the protein expression levels of GP4,GP6,GP1 BB,ITGA2B and SELP in HA rats were significantly higher than that of SE rats(P<0.05).The expression levels of PF4 and β-TG in PPP of HA rats were higher than that of the SE group rats.Functional experiments show that chronic hypoxia exposure can induce platelet activation in rats,and the bleeding time of rats in the HA group was found to be significantly shorter than that of SE group rats(P=0.000).The spreading area of platelets in the HA group was significantly larger than that of the SL group(P=0.0002).The clot area of the HA group was significantly smaller than that of the SE group,regardless whether the duration was 30 min(P=0.004)or 120 min(P=0.002).Conclusion: Chronic hypoxia exposure at high altitudes decreased platelet count,causes phenotypic changes,alters gene profile,membrane receptors(GP4,GP9,GP6 ITGA2 B,SELP,GP1BB)and chemokine(PF4V1)expression increases.The platelet ultrastructure changes,triggers the release of alpha granules,PF4,β-TG and ADP release increases,while functional experiments found platelet spreading and clot retraction enhancement,which may be the main cause of thrombosis that results from chronic hypoxia exposure.