MP055C5B9 DEPOSITS ON ENDOTHELIAL CELLS FOR THE EVALUATION OF COMPLEMENT FUNCTION IN THROMBOTIC MICROANGIOPATHIES OF DIFFERENT ORIGIN & THERAPY MONITORIZATION

Abstract

Background: Atypical hemolytic uremic syndrome (aHUS) is a rare, progressive, life-threatening form of thrombotic microangiopathy (TMA) caused by dysregulation of the alternative pathway of the complement system. Nonetheless, there is wide evidence that complement activation has a role in other TMA. Aims: A reliable method is needed to evaluate complement activation to monitor eculizumab therapy in aHUS and to explore the indication of this treatment in other TMA. Methods: Complement activation was assessed by exposing endothelial cells (ECs) to patients' sera or to patients' plasma samples mixed with a control sera pool (1:1). C5b9 deposits on ECs were analyzed through immunofluorescence and expressed as fold increase (mean±SEM) vs. control samples . Results: Exposure of ECs to aHUS plasma resulted in a significant increase in C5b9 deposits (8±2, n=4, p< 0.01), which was prevented in samples from the same patients treated with eculizumab (0.7±0.1). Significant fibrin formation was observed together with C5b9 deposition. The specificity of the reaction was confirmed by its blockade with eculizumab and inhibitors of the intrinsic coagulation pathway. Notably, results obtained using plasma samples were much more remarkable and reproducible than those obtained with sera. C5b9 deposition was also increased using samples from HELLP syndrome patients (at acute phase, 6±2, n=4, p< 0.01) and 40 days later (3.2±1, p< 0.01), and both autoimmune (2.9±1, n=4, p< 0.05) and congenital thrombotic thrombocytopenic purpura (2.1±1, n=4). Complement activation was at control levels when analyzing samples from patients with malignant hypertension (0.8±0.2, n=5) or aHUS patients treated at regular doses of eculizumab (0.5±0.1, n=5) and lower (0.4±0.2, n=2). Conclusions: This method could be useful to monitor therapy and to explore the indication of eculizumab to treat other TMA presenting complement activation. Further research is needed to clarify the basis for the colocalization of C5b9 and fibrin on ECs surface.