The Influence Of Annexin A5on The Thrombophilia Of Systemic Lupus Erythematosus

Background and Objective:Systemic lupus erythematosus (SLE) is a classic autoimmune disease. As the short-termsurvival rate of SLE increased in these decades, chronic patients now suffer from increasingthrombosis-related symptoms, which had become a significant lethal factor for long-termprognosis. Our team used comparative proteomic strategies to identify16disregulatedproteins from peripheral blood monouclear cells (PBMCs) of SLE patients. Annexin A5(AnxA5) is one of the upregulated proteins, which we thought we should pay attention to.AnxA5is a distinctive annexin with anticoagulant activity that is rarely observed in theannexin family, but its detailed pathogenesis in SLE remains unclear. If the detailed functionof AnxA5in SLE could be elucidated, it should be very meaningful to help the SLE patientsto avoid the serious vascular events. To provide further insight about AnxA5into this disease,firstly we proposed to start our study about the AnxA5expression in peripheral blood and theclinical manifestation correlation. Secondly, we determined the sera concentrations of serveralanti-phospholipid antibodies, and analyzed their associations between AnxA5sera level ordifferent clinical parameters. At last we applied the recombination AnxA5and anti-AnxA5monoclonal IgG to intervene the progress of several blood coagulation screens, in order toprobe the possible mechanism pathway of the influence of AnxA5on lupus-relatedthrombophilia.Methods:The study involved200SLE patients and174healthy women.1. A proteomic analysis was accomplished using two-dimensional electrophoresis (2-DE)and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) from PBMCslysate. Protein identification and expression were validated using Western blot.Semi-quantitation of bolt film bands were executed with Quantity One software and statisticalcorrelations between clinical manifestation were analyzed with SPSS software or manually, when necessary.2. Sera AnxA5levels and four potentially related molecules, anti-AnxA antibodies, β2glycoprotein I (β2GPI), anti-oxidized phospholipids (anti-Ox-PLs) and anticardiolipin IgG(aCL IgG) were determined using enzyme-linked immunosorbent assays (ELISA). Statisticswere also analyzed with SPSS software.3. Recombination AnxA5and anti-AnxA5monoclonal IgG were used as intervenors andbovine serum albumin (BSA) were use as unrelated control. Preliminary parallel coagulationassays of SLE patients and healthy controls were processed using an automatic analyzer.Results:1. Western blotting confirmed the over-expression of AnxA5, from the PBMCs of themost SLE patients including (SLE vs. control=60.7%vs.6.4%, P=0.0004).2. No significant difference was observed in the results from the screening of thecytomembrane protein lysate. On the other hand, over-expression of AnxA5from theintracelluer protein lysate was observed from the SLE patients comparing to healthy control.3. Reviewing the correlations indicated the cluster without cyclophosphamide (CTX)treatment exhibited much higher AnxA5than the CTX-treated cluster (treated vs. untreated=17.0%vs.97.4%, P=0.014, without dose-dependence).4. ELISAs demonstrated the AnxA5concentrations of the patient sera (26.8±3.0ng/mL)were generally lower than the healthy donors (49.0±3.3ng/mL, P=0.0001), except forseveral patients, whose sera AnxA5were similar with, even higher than the most healthydonors.5. A positive correlation was demonstrated between the manifestation of thrombosis andAnxA5(Mann-Whitney Z=-2.084,P=0.037).6. No significance were found when searching for correlations between AnxA5and theother4molecular levels, anti-AnxA5, β2GPI, anti-Ox-PLs and aCL IgG.7. The correlation between anti-AnxA5and lupus-related thrombophilia was notcompletely certain, but this disputed statistic was mostly contributed the anti-AnxA5extremum values (Pearson's (r)=0.340,P <0.0005vs. Mann-Whitney=-1.272,P=0.203).8. The coagulation assays using sera from SLE patients revealed that both elevatedAnxA5and anti-AnxA5shortened prothrombin time (PT), activated partial thromboplastintime (APTT) and prolonged thrombin time (TT)(P <0.01). 9. Anti-AnxA5raised functional fibrinogen levels of sera from SLE patients.10. The intervention of AnxA5in healthy control group displayed a similar trend to theSLE groups on PT, APTT and TT, but the alterations were not as numerically significant orstatistically dramatic as these in SLE.Conclusions:1. The heterogeneous transcellular distribution, increased intracellular concentrations anddecreased serum levels of AnxA5represent a protective response to lupus-relatedthrombophilia.2. AnxA5rarely correlated with several aPLs mentioned above, and could probably bean independent factor in the pathogenesis of SLE.3. In the thrombogenesis of SLE, AnxA5and its antibodies, might mostly participate inthe common coagulation pathway, which promoted the hypercoagulation state of SLE.