Clusterin can mediate apoptosis-induced molecular mechanisms in immune thrombocytopenia

• Platelet and megakaryocyte apoptosis may be involved into the pathogenesis of ITP through a mechanism associated to CLU and its regulators, GRP78 and GRP94, potentially by activating Bax

Abnormalities in the apoptosis pathway have been implicated into the pathophysiology of various autoimmune diseases, including immune thrombocytopenia (ITP). Platelets and megakaryocytes (MKs) may be seen as the major targets for the pathogenic immune responses in ITP. Our data suggests that mechanisms associated with impaired clusterin-mediated apoptosis might play a role in the pathophysiology of ITP platelets and their production by MKs. Platelet-rich plasma (PRP) from 10 ITP patients compared to healthy controls was used for the apoptosis proteomic profiling and clusterin (CLU) expression validation by RT-qPCR. We used the MEG-01 cell line, treated for 2h with plasma from newly diagnosed (ND), chronic ITP patients or healthy controls and pan-caspase inhibitors (Z-VAD-FMK), apoptosis inducer ABT-737, Rotenone or Rapamycin. Our apoptosis proteomic profiling revealed significantly increased expression levels of certain apoptotic genes such as CLU, Phospho-p53 (S46), Procaspase-3, and cleaved Caspase 3 (CASP3) in ITP patients compared to healthy controls. Treatment with pan-caspase inhibitor or Rapamycin had a significant downregulatory effect at mRNA level for CLU, CASP3, -8 and -9, p53, and BCL-2-associated X protein (BAX) in ITP plasma treated cells in comparison to control plasma treated MEG-01 cells, whereas Rotenone or ABT-737 had opposite effects. We observed a significant downregulation of mRNA expression levels of these apoptotic markers in ITP plasma treated and CLU or GRP78 siRNA transfected MEG-01 cells. Our results indicate a possible impairment of apoptosis pathway via upregulation of CLU and BAX in platelets and in their producers MKs that may contribute to platelet destruction in ITP disease.