Study Of Citrullinated Proteins In Platelets And Platelet Derived Microparticles Serve As Autoantigen For ACPA Recognition

Rheumatoid arthritis(RA)is a chronic autoimmune disease characterized by persistent systemic inflammation,synovial hyperplasia and erosive bone destruction,and is difficult to cure.Early and accurate diagnosis is the key to avoiding serious joint damage and complications in patients with RA.Anti-citrullinated protein antibodies(ACPA)is an important serological diagnostic mark in the early stages of RA,which have great reference value for disease progression and prognosis.ACPA targets a variety of citrullinated proteins catalyzed by peptidyl-arginine deiminase(PAD),and has high specificity for RA.Revealing the potential targets of ACPA is important for understanding disease pathogenesis and diagnosis.Several studies have demonstrated elevated platelet-activated aggregation in the blood of RA patients and the detection of platelet derived microparticles(PDPs)fractions in the joints.Which citrullinated proteins are present in platelets and PDPs,how these citrullinated proteins are produced,and whether they can be used as recognition targets for ACPA in RA disease to induce ACPA production remains to be investigated.In this study,the expression of PAD isozyme m RNA and protein in platelets was detected using RT-PCR and in-gel enzymatic digestion bridged to LC-MS/MS,respectively.Then,citrullinated proteins and modification sites in platelets and PDPs were identified through FASP enzymatic hydrolysis and high-precision LC-MS/MS,ACPA was detected by ELISA on citrullinated TSP-1,β-actin and PF-4 by ELISA,and ACPA was shown to induce platelet activation by the detection of platelet activation markers CD62 P and s CD40 L.The main findings were as follows:(1)Expression of PAD4 m RNA and protein was detected in platelets,and PAD4 was the only PAD isoenzyme detected in platelets at both m RNA and protein levels.(2)A total of 44640 unique peptides and 3973 proteins were identified in platelets,and Maxquant unlabeled quantitative analysis showed that platelet protein abundance spanned ten orders of magnitude,with the most abundant protein being β-Actin,accounting for 4.0% of the total protein content.GO functional enrichment analysis of platelet proteins showed that the most abundant proteins were involved in the organization of the cytoskeleton,the release of alpha particles and the signal transduction processes in which the cytoskeleton is involved.(3)117 citrullinated sites on 83 proteins were identified in platelets,among which proteins with more citrullinated sites were fibrinogen α,integrin αIIb,myosin 9 and TSP-1.The main biological processes involved in platelet citrullinated proteins include cytoskeletal tissue,platelet particle release,membrane signal transduction,Golgi vesicle transport,antigen processing,coagulation,and protein processing.(4)In PDPs,5477 unique peptides and 688 proteins were identified,of which 37 proteins underwent citrullination modification at 55 sites.15 citrullinated proteins such as β-actin,filamin,and integrin αIIb were detected in both platelets and PDPs.(5)Compared with native TSP-1,β-actin and PF4,citrullinated TSP-1,β-actin and PF4 were highly immunogenic and bound to ACPA in RA serum.(6)Incubation of RA serum,SF and ACPA with platelets significantly increased platelet CD62 P expression and s CD40 L secretion,suggesting that ACPA can effectively induce platelet activation after targeting binding to citrullinated proteins in platelets.This study comprehensively reveals the citrullinated proteome in platelets and PDPs and identifies that platelet citrullinated proteins can act as autoantigens recognized by ACPA,and ACPA binding to citrullinated proteins promotes platelet activation.Our findings suggest that the presence of citrullinated autoantigens in platelets and PDPs plays an important role in the pathogenesis of RA.