The Role Of Gly14-Humanin(HNG)in Platelet Activation And Thrombus Formation

Background:Cardio-cerebrovascular diseases(CVD)are the leading causes of mortality and disablility worldwide.With limited understanding of the pathogenesis,early diagnosis,effective treatment,and prophylaxis of CVD are still a major challenge of health care.Platelet activation is essential to thrombus formation,which is a priming step involved in cardiovascular events including myocardial infarction and stroke Therefore,developing effective anti-platelet drugs remains a major task in controlling CVD.HNG,a homologue of humanin with enhanced anti-apoptotic activity,confers protection against myocardial ischemic-reperfusion damage,ischemic stroke,and atherosclerosis in addition to the amelioration of amyloid β-related AZD pathological changes.However,whether HNG affects platelets,a common mediator of CVD,is still unknown.Therefore,we hypothesized that HNG may also inhibit platelet function and thrombus formation.This study will evaluete the role of HNG in platelet activation and thrombus formation and shed light on developing novel anti-platelet agentsAims:1.To test the role of HNG in platelet activation2.To identify the drug target of HNG in platelets3.To aveluate the anti-thrombotic role of HNG.4.To test the role of HNG in thrombo-inflammation induced by myocardial ischemia-reperfusion(I/R)Methods:1.Determination of the role of HNG in platelet activation(1)Platelet aggregation:Gel-filtered human platelets were incubated with HNG or vehicle for 10 minutes,stimulated with various agonists(collagen,CRP,thrombin,AYPGQV)for 5 minutes,and aggregation was assessed using an aggregometer.(2)Platelet granule release:ATP release was measured using an aggregometer and P-selectin expression was detected by flow cytometry.(3)Inside-out and outside-in signaling pathways:Inside-out:αⅡbβ3 activation was tested by measuring membrane-bound soluble fibrinogen using flow cytometry.Outside-in:Pretreated platelets were allowed to spread on immobilized fibrinogen and stained with phalloidin to calculate platelet spreading areas.(4)Platelet signaling pathway:Western blotting was used to detect the intracellular kinase activation.2.Assessing the role of HNG in thrombosis and hemostasis.(1)Flow chamber:Platelet adhesion under shear was measured using a BioFlux200TM flow chamber system.Channels in BioFluxTM plates were primed,coated with type I collagen(200 μg/ml)for one hour at RT.Human whole blood anticoagulated with sodium citrate was preincubated with HNG or vehicle,labeled with Calcein-AM,and perfused in the channels at 10dyn/cm2.Adherent platelets were quantified by fluorescence recorded under an inverted microscope.(2)Ferric chloride induced carotid artery injury model:Mice were pretreated intrapcritoncally with HNG(25 μg/kg)(n=13)for 60 min,then the left carotid artery was dissected,soaked in 7.5%FeCl3 for two minutes.Time to occlusion of the carotid artery was determined as the duration from FeCl3 stimulation to the loss of blood flow.(3)Tail bleeding test:Mice were anesthetized,the distal 3 mm of the tail was transected,and immediately immersed in 12 ml of 0.9%sodium chloride for 30 min at 37℃.The first time to bleeding cessation and the re-bleeding time were recorded.3.Evaluation of the role of HNG in myocardial ischemia reperfusion(I/R)induced thrombo-inflammation:(1)Model of myocardial ischemia-reperfusion injury in mice:HNG(0.2mg/kg)or saline was administrated o mice,then the operation and sham operation were performed.After ischemia for 45 minutes and reperfusion for 10 minutes or 2 hours,whole blood or heart were obtained for further experiments.Evans Blue and TTC staining were performed for determining myocardial infarct size.The unstained blue areas of the myocardium(risk area,AAR),Evans Blue stained blue areas(non-hazardous area,ANAR),TTC stained areas(red)and TTC unstained areas(infarct area,IA,white)were measured using Image J software.Myocardial infarction size is the percentage of infarcted area to dangerous area(AAR),while the AAR is the ratio of AAR to total left ventricular area(AAR/AAR+ANAR).(2)The effect of HNG on thrombo-inflammation induced by myocardial ischemia/reperfusion injury.The expression of P-selectin,reactive oxygen species production and the platelet-leukocyte aggregates were measured using flow cytometry.4.Identifying the target of HNG in platelets.(1)Screening the potential targets of HNG in platelets:Avidin and biotin affinity purify and mass spectrometry were used to screen the potential HNG-binding proteins on platelets.Bioinformatics analysis was performed,including KEGG pathway,GO function and protein interaction(PPI)analysis.(2)Molecular docking:Protein structure models for HNG(2GD3)were downloaded from the RCSB PDB database(http://www.rcsb.org/).The putative humanβ-1 tubulin structure was constructed using the ModBase server based on the crystal structure of human β2-tubulin(RCSB PDB:4I4T).In silico protein-protein interaction was predicted using the ZDOCK Server(http://zdock.umassmed.edu/).Docking results were evaluated using the FibcrDock Server(http://bioinfo3d.cs.tau.ac.il/FibcrDock/).(3)Immunoprecipitation and immunofluorescence experiments:Biotin-avidin affinity purify and immunoblot Assay:Biotin-labeled HNG was incubated with platelet lysate,and purified using avidin beads.Then the proteins were seperated by SDS-PAGE,and blotted with β-tubulin antibody.Immunofluorescence Assay:After incubation with FITC-labled HNG,platelet microtubules and F-actin proteins were stained with β1-tubulin and phalloidin,respectively,and pictures were photographed using laser confocal.Microtubule depolymerization assay:HNG-pretreated platelets were incubated at 37℃ with nocodazole.Alternatively,platelets were allowed to adhere to fibrinogen-coated slides before fixation.Samples were stained using an anti-β1-tubulin and anti-acetylated tubulin antibody.Results:1.HNG inhibits platelet activation.(1)HNG inhibits platelet aggregation:HNG dose-dependently inhibits collagen-induced platelet aggregation with a 58%reduction(P<0.01,n=8 for each group).HNG also inhibited platelet aggregation induced by convulxin(P<0.01),,AYPGQV(P<0.05),and thrombin(P<0.05)(2)HNG inhibits platelet P-selectin expression and ATP secretion:HNG significantly inhibited the augmentation of P-selectin expression induced by convulxin compared with vehicle(P<0.01).Platelets treated with HNG showed significantly lower ATP release induced by collagen(P<0.05)and convulxin(P<0.05)compared to those treated with vehicle(3)HNG inhibits platelet integrin αⅡbβ3 activation and spreading on fibrinogen:Compared with vehicle,HNG significantly decreased the level of platelet surface fibrinogen induced by convulxin(P<0.01).HNG significantly decreased platelet spreading area compared with vehicle(P<0.05)(4)HNG inhibits Akt/Erk1/2 signaling in platelets:Phosphorylation of AKT and ERK1/2 induced by collagen was significantly decreased by HNG.Phosphorylation of p38MAPK was mildly decreased by HNG.2.HNG inhibits thrombus formation in vivo without increasing bleeding time(1)HNG inhibits platelet adhesion on collagen in flow conditions:Live fluorescence imaging showed that HNG significantly decreased platelet adhesion on collagen surface compared with vehicle(P<0.01)(2)HNG inhibits thrombosis in vivo:Ultrasonographical observation of carotid blood flow showed that HNG-treated mice had longer initial occlusion time(7.3±0.4 min,n=10)compared with the control group(5.4±0.7 min,n=12)(P<0.05).Ultrasound examination of carotid blood flow showed that the initial occlusion time of thrombus(P<0.05)was prolonged after intraperitoneal injection of HNG(25 μg/kg,n=13)in mice(3)HNG did not increase tail bleeding time:Compared with the control group,no prolongation of bleeding time was observed in HNG-treated(25μg/kg)mice3.HNG reduces thromboinflammation after myocardial I/R:We found that HNG reduced mouse heart infarct size after I/R(P<0.05).Besides,we also found that HNG reduced the expression of P-selectin,ROS production and platelet-leukocyte complex(P-selectin,P<0.01;ROS production,P<0.01;platelet-leukocyte aggregates,P<0.05)4.HNG may bind to platelet β1-tubulin and stabilize microtubules.(1)Mass spectrometry analysis revealed β1-tubulin as one HNG-binding protein in platelets.(2)Molecular docking suggested that HNG binds to β1-tubulin at a distinct site from the known binding-pocket to taxol.(3)Biotin-avidin affinity purification pull down showed that HNG was co-immunoprecipitated with platelet β1-tubulin.(4)HNG was colocalized with β1 tubulin in platelets.(5)HNG inhibited platelet microtubule contraction and depolymerization induced by fibrinogen or nocodazole.Conclusions:1.Our study showed that HNG inhibits platelet activation,arterial thrombosis without increasing bleeding time.2.Our results showed that HNG may attenuate thromboinflammation induced by myocardial ischemia reperfusion.3.HNG may bind to platelet β1-tubulin and prevent microtubule depolymerization and contraction.4.Our study suggested that HNG has a protective role in myocardial ischemia-reperfusion injury.It provide a new mechanism for the development of HNG as an antiplatelet and antithrombotic drug in future.