Up-Regulation of the mRNA Expression and Plasma Activity of PADI4in Sickle Cell Anemia during Acute Crisis

Introduction: Recent evidence suggests that coagulation activation is involved in the pathogenesis of progressive organ failure in Sickle Cell Anemia (SCA). In addition, generation of neutrophil extracellular traps (NETosis), one of the components of immunothrombosis, has been associated with the pathogenesis of both venous thromboembolism and SCA. NETosis is a complex process that involves the orchestrated participation of several proteins such as peptidyl arginine deaminase (PADI4), neutrophil elastase (ELANE) and myeloperoxidase (MPO). PADI4 mediates histone citrulination, an essential step for NETosis. Accordingly, its inhibition has been recently cited as a potential therapeutic strategy for diseases in which NETosis are thought to play a relevant pathogenic role such as SCA. Although attractive, investment in PADI4 inhibitors in SCA requires gathering of more convincing evidences of the role of this enzyme in its pathogenesis. Herein, we used two cohorts of patients to investigate the expression of NETosis regulators (PADI4, ELANE and MPO) in SCA at steady state and during acute crisis, and to assess whether PADI4 activity is increased in any of these states. Methods: patients were recruited from two different centers in Brazil. Whole blood samples were obtained from patients from cohort 1 at steady state or during acute crisis (within 24 hours from admission). mRNA was obtained from granulocytes isolated by Ficoll gradient and gene expression of PADI4, ELANE, and MPO were measured by qPCR. In patients from cohort 2, PADI4 and MPO activity were measured in samples obtained within 24 hours from admission and after patient discharge (convalescence) using commercial kits in serum (MPO, Myeloperoxidase activity assay kit; ab105136) and plasma (PADI4, PAD4 Inhibitor Screening Assay Kit; Cayman chemical). Healthy individuals from the same geographic region were used as controls for each cohort, independently Results: In total, 54 steady state patients, 27 acute crisis and 40 healthy volunteers were evaluated for mRNA expression of NETosis regulators. Patients in acute crisis expressed higher levels of PADI4, MPO and ELANE compared to both healthy volunteers and patients in steady-state. Furthermore, plasma activity of PADI4 was higher in acute crisis when compared to healthy individuals (7.36x10⁶ vs 5.24x10⁶; P= 0.002), with no decrease after discharge (median of 13.5 days after admission) (7.36x10⁶vs 7.41x10⁶; P= 0.004). No differences were observed in serum MPO activity during acute crisis. Conclusion: we demonstrate that the mRNA expression of NETosis regulators, including PADI4, is increased in SCA; and that this increase is associated with higher levels of PADI4 activity in plasma during acute crisis. These results support the concept of PADI4 inhibition as a therapeutic strategy for acute episodes of SCA, and warrant additional studies in this area.

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