Clusterin can mediate apoptosis-induced molecular mechanisms in immune thrombocytopenia

Abstract

Abnormalities in the apoptosis pathway are possible risk factors for various autoimmune diseases including immune thrombocytopenia (ITP). ITP is an autoimmune bleeding disorder characterized by a low platelet count and mostly mild but in rare occasions life threatening bleeding symptoms. Platelets and megakaryocytes (MKs) may be seen as the major targets of the pathogenic immune responses in ITP. A mechanistic understanding of the ITP pathogenesis is still lacking. Our data indicate that mechanisms associated with impaired clusterin-mediated apoptosis might play a role in ITP platelet pathophysiology and platelet production by MKs. We could demonstrate by apoptosis proteomic profiling significantly increased expression levels of some apoptotic genes such as clusterin (CLU), pro-caspase 3, catalase, TRAILR1/DR4, Bax, Bad and Bcl-2 compared to healthy controls in platelet-rich plasma (PRP) from 10 ITP patients. We could validate by both RT-qPCR and Western blotting that CLU, a stress-activated chaperone, is significantly increased in both newly diagnosed and chronic ITP. We used the human megakaryoblastic cell line MEG-01, treated for 4h with plasma from acute and chronic ITP patients and healthy controls. We performed chemical treatments in plasma treated MEG-01 by using pan-caspase inhibitors (Z-VAD-FMK), apoptosis inducer ABT-737, Rotenone and Rapamycin. We determined the expression at mRNA levels of apoptosis pathway regulatory genes Bax, caspase-3, -8, -9 as well as CLU, GRP78 and GRP94 by qRT-PCR. We could demonstrate significantly downregulation of mRNA expression levels of these apoptotic markers in ITP plasma treated and CLU 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 can lead to platelet destruction in ITP disease.