The Role Of Secretory Protein Disulfide Isomerases ERp57 And ERp72 In Platelet Aggregation And Thrombosis,and The Underlying Mechanisms

Background:Thrombosis is a major cause of arterial thrombotic diseases leading to myocardial infarction and stroke which are life threatening disorders.According to a joint investigation reported by Lancet in 2017,cardiovascular disease is the most common cause among the population over 40 years old.However,there are still lack of effective strategy for prevention and treatment of these diseases.Thus,understanding the mechanisms of thrombosis is critical for elucidation of the pathogenesis of thrombotic diseases.Recent studies demonstrate that protein disulfide isomerase(PDI)is critical for both platelet activation and thrombosis.A group of proteins having same function with PDI is a family of enzymes catalyzing disulfide bond formation,reduction and isomerization in the endoplasmic reticulum.Human PDI family contains several members,including PDI,ERp57 and ERp72,etc.It have been shown that several PDI members act distinctly and coordinately to regulate the physiological function in mammalian cells.Endoplasmic reticulum protein 57(ERp57)is a member of the PDI family.Our study using ERp5 7 inhibitors has suggested that ERp5 7 is important for platelet aggregation,hemostasis and thrombosis.The physiological role of ERp57 in platelet activation and thrombosis,however,is still unknown.Whether and how ERp57 regulates the function of integrin αⅡbβ3 is another important question remaining to be answered.The reductase activity of the N-and C-terminal active sites are not equivalent,but nothing is known about the relative importance of each active site in platelet function.Endoplasmic reticulum protein 72(ERp72)is one of the largest PDI family member that contains an additional CGHC active site at its N terminal.ERp72 shares high sequence identity with ERp57 and is also secreted from platelets upon activation.However,the role of ERp72 in platelet activation is totally unknown.Aims:Our first aim is to determine the physiological role of platelet-derived ERp57 in platelet activation and thrombosis and the underly:ing molecular mechanism.The second aim of this study is to evaluate the role of ERp72 in platelet aggregation and thrombosis.We also compared ERp72 and ERp57 in terms of their distinct roles in regulation of platelet activation.Methods:In this study,to determine the role of platelet-derived ERp57 in platelet accumulation at the site of injury and the underlying mechanism,we used platelet-specific ERp57 knock-out mice(PF4-Cre/ERp57-/-),integrin β3 knock-out mice and αⅡbβ3 inhibitor eptifibatide,as well as intravital microscope and ferric chloride(FeCl3)-induced mesenteric arteriole thrombosis model.To determine whether ERp57 binds to αⅡbβ3 on platelets,we used flow cytometry to compare the binding of Alexa 488-labeled ERp57 protein between wild type and β3 knock-out platelets.Sulfhydryl labeling was used to study the role of ERp57 in thiol-disulfide exchange in αⅡbβ3 molecules.We also determine the functional site of ERp57 in platelet aggregation using inactive mutants of ERp57-CGHC domains.To study the role of ERp72 in platelet aggregation,we prepared specific anti-ERp72 inhibitory antibody and ERp72 inactive site mutants and tested them in platelet aggregation and ATP secretion.We used ERp72 knock-out mice(Tie2-Cre/ERp72-/-)to study the role of ERp72 in thrombosis in ferric chloride(FeCl3)-induced mesenteric arteriole thrombosis.We also performed MPB labeling assay of αⅡbβ3 for better understanding the mechanism by which ERp72 regulates disulfide reduction in this molecule.Mass spectrometry was used to identify new free thiols generated during αⅡbβ3 activation.Results:(1)PF4-Cre/ERp57-/-mice displayed prolonged tail bleeding times and thrombus occlusion times in ferric chloride(FeCl3)-induced carotid artery model.(2)Using a mesenteric arteriole thrombosis model,we found a decrease in incorporation of ERp57-deficient platelets into a growing thrombus.Since αⅡbβ3 is critical for this incorporation,our result indicates ERp57 regulates platelet accumulation in anαⅡbβ3-dependent manner in vivo.(3)αⅡbβ3-mediated platelet function,includingαⅡbβ3 activation,platelet spreading and clot retraction were inhibited by ERp57 deficiency.(4)Surface expression of ERp57 protein and activity in human platelets increased with platelet activation.(5)Binding of Alexa 488-labeled ERp57 to thrombin-activated and Mn2+-treated platelets lacking β3 was decreased substantially,suggesting a direct interaction of ERp57 with αⅡbβ3.(6)ERp57 mutant containing a functional C-terminal CGHC active site potentiated platelet aggregation,indicating the C-terminal CGHC active site is critical for platelet aggregation.(7)ERp57 wild type,as well as the mutant containing a functional C-terminal active site generate sulfhydryls inαⅡbβ3 molecules,indicating ERp57 and its C-terminal active site is important in disulfide reduction in allbp3.(8)We generated a polyclonal anti-ERp72 antibody which specifically recognized recombinant ERp72 and ERp72 in human and mouse platelets,and inhibited the reductase activity of ERp72.This antibody inhibited platelet aggregation and ATP secretion in a dose dependent manner,indicating that ERp72 is important in platelet activation.(9)ERp72 mutants containing both of the second and third active sites potentiated platelet aggregation.ERp72 mutants containing the second and third single active sites had similar effects on aggregation,suggesting that the second and third active sites of ERp72 are equally important.(10)Recombinant ERp72 bound to integrin αⅡbβ3 on platelet surface,and the second and third active sites generated free thiols in αⅡbβ3 in a dose-dependent manner.(11)Intracascular ERp72 regulates platelets accumulation in vivo.(12)ERp72 binds to integrin αⅡbβ3 on platelet surface.(13)When added to ERp57-null platelets,recombinant ERp57 recovered the defect of aggregation whereas recombinant ERp72 failed to do this,indicating that ERp57 and ERp72 play distinct role in aggregation.(14)In a purified system,ERp57,PDI and ERp72 showed different activities on generation of free thiols in αⅡbβ3 molecules.(15)New free thiols were generated during αⅡbβ3 activation as detected by mass spectrometry.Conclusions:Platelet-derived ERp57 is necessary for thrombus formation,platelet accumulation at the site of injury and integrin αⅡbβ3-mediated platelet function.ERp57 binds to platelet surface mainly through integrin αⅡbβ3 and regulates disulfide reduction in αⅡbβ3.The C-terminal CGHC active site of ERp57 is important in platelet aggregation and disulfide reduction in αⅡbβ3.Intravascular ERp72 is critical for regulation of platelet activation in vivo.The second and third active sites of ERp72 are important for aggregation and ATP secretion.ERp72 binds to αⅡbβ3 and generates free thiols by its second and third active sites.ERp57 and ERp72 act separately in platelet aggregation and have distinct effects on free thiol generation in αⅡbβ3.