Complement C5a Induces The Generation Of Neutrophil Extracellular Traps By Inhibiting Mitochondrial STAT3 To Promote The Arterial Thrombus

Background and PurposeArterial thrombosis is the cause of myocardial infarction(MI)and stroke,which account for the major burden of mortality and disability from cardiovascular events globally.Antithrombosis therapy is the main prevention and treatment but has an inherent risk of bleeding complications.Inflammation and thrombosis are tightly connected processes,contributing to the containment of pathogens via a host defense effector mechanism termed "immunothrombosis".Due to dysregulation of immunothrombosis,there is likely a key event in the development of thrombotic disorders,including myocardial infarction and stroke.Thrombotic events cannot be completely prevented by antithrombotics,implicating a therapeutic gap due to inflammation,a not yet sufficiently addressed mechanism.Inflammation was originally characterized by an immune response to pathogenic organisms,where neutrophils are the first cells of the immune system to migrate to and form extracellular chromatin nets decorated with histones and numerous granular proteins to trap bacterial pathogens,namely,neutrophil extracellular traps(NETs).Considerable recent evidence implicates NETs as a major player in thrombosis,for example,providing scaffolds for palates and erythrocytes,assistantly activating factor Ⅻ,and stabilizing fibrin nets.NETs are an essential interface between inflammation and thrombosis.There are some reports on targeting NETs for antithrombotic therapy,showing a reduced thrombus burden.DNase,an enzyme that attacks DNA,which serves as the backbone of NETs,and chloroamidine,an inhibitor of peptidylarginine deiminase 4(PAD4)that has been shown to be critical for NET release,are the main focus of targeting NETs.However,histones and neutrophil elastase(NE)remain after digestion by DNase in NETs and still can promote thrombosis.PAD4 inhibition may attenuate the antibacterial innate immunity mediated by NETs,and PAD4 does not play an integral role in NADPH oxidase(Nox)-dependent NET formation.Various triggers,such as cytokines,bacterial components,the experimental agonist phorbol-12-myristate-13-acetate(PMA),or activated platelets,can stimulate the activation of neutrophils,which leads to the concomitant release of NETs into extracellular compartments.Initiators that trigger NETosis originally in thrombosis could be new options for targeting NETs.However,exactly how the NETotic process is initiated and maintained during arterial thrombosis remains incompletely understood.Several studies suggest that complement C5a is closely related to thrombosis.In vitro,neutrophil chemotaxis is enhanced by complement c5a in coronary thrombus-derived plasma.In vivo,neutrophils accumulate at the site of thrombus formation and are correlated with C5a and enzymatic infarct size.In a deep vein thrombosis(DVT)model,both thrombotic stability and load are reduced in C5-deficient mice.In sepsis,C5a stimulates neutrophils to produce NETs.Whether C5a is the major molecule that activates neutrophils to produce NETs,thus promoting the occurrence and development of thrombosis,has not yet been demonstrated.This study was initiated to investigate the relationship between C5a and NETs in arterial thrombosis.Methods1.The expression of C5a in plasma and NETs in thrombus:The concentrations of C5a in plasma of STEMI patients were higher than that of angina pectoris patients,and both C5a and NETs were higher in FeC13 induced arterial thrombosis model group than that in sham operation group.2.Compared with NaCl group,PMX53 group reduced the areas and weights of arterial thrombus,retained more internal carotid blood flow,and also reduced the expression of NETs in thrombosis.In vitro,C5a stimulated neutrophils to produce NETs.3.C5a increased the production of mitochondrial ROS and that the formation of NETs promoted by C5a was mitochondrial ROS dependent.Inhibition of mitochondrial ROS generation with MitoTEMPO reversed the ability of C5a to facilitate the release of NETs.4.Compared with that in the control group,the ratio of mitochondrial p-STAT3(Ser727)to mitochondrial total STAT3 was lower after stimulation with C5a.An inhibitor of the STAT3 pathway,AG490,promoted the production of NETs.5.Through pharmacologic blockade of the mitochondrial STAT3 pathway,AG490 abolished the reduction in arterial thrombotic burden induced by PMX53 in vivo.The reduction in thrombus weight induced by PMX53 was suppressed by AG490.In addition,the reservation of more blood flow in the LICA by PMX53 was reversed by AG490.There were fewer NETs in thrombi after injection of PMX53,which was reversed by AG490.6.In the presence of the STAT3 agonist Colivelin,the effect of C5a on the increased production of Mito-ROS was suppressed.In addition,the application of Colivelin reversed NETosis induction by C5a,indicating that members of the Mito-STAT3 signaling pathway are key mediators of the process of C5a-induced NETosis.ConclusionThe concentrations of C5a in plasma of STEMI patients were higher than that of angina pectoris patients.By inhibiting mitochondrial STAT3 to elicit mitochondrial ROS generation,which facilitates the NETotic process,C5a triggers the generation of NETs to promote the development of arterial thrombosis.Hence,our study identifies complement C5a as a potential new target for the treatment and prevention of thrombosis.