PB0730 - Alterations in apoptosis and autophagy/ER-phagy signaling pathways contribute to the pathogenesis of immune thrombocytopenia

Monday, June 24, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

CB

Christina Bitsina, Msc

PhD Student
University Children’s Hospital Zürich, Division of Hematology, University of Zürich, Zürich, Switzerland
Zürich, Zurich, Switzerland

Co-Author(s)

TS

Tea Stein

Doctor
University Children’s Hospital Zürich, Division of Hematology, University of Zürich, Zürich, Switzerland
Zürich, Zurich, Switzerland
SS

Stacey Shllaku

Graduate Student
University Children’s Hospital Zürich, Division of Hematology, University of Zürich, Zürich, Switzerland
Zürich, Zurich, Switzerland
NG

Nadine Gölz

Dr.
University Children’s Hospital Zürich, Division of Hematology, University of Zürich, Zürich, Switzerland
Zürich, Zurich, Switzerland
MS

Markus Schmugge

Prof. Dr. med.
University Children’s Hospital Zürich, Division of Hematology, University of Zürich, Zürich, Switzerland
Zürich, Zurich, Switzerland
FF

Francesca D. Franzoso

Doctor
University Children’s Hospital Zürich, Division of Hematology, University of Zürich, Zürich, Switzerland
Zürich, Zurich, Switzerland

Background: Immune thrombocytopenia(ITP) is an autoimmune disorder characterized by low platelet counts. Previous studies, including our group, revealed some increased apoptotic markers in platelets and megakaryocytes (MKs), but the mechanisms remain unknown.

Aims: We aimed to investigate the gene expression and activation mechanisms of apoptosis and autophagy/ER-phagy in MKs.

Methods: We used the MEG-01 cell line treated either with pan-caspase inhibitor (Z-VAD-FMK), Rotenone, Rapamycin, ABT737 or transfected with siRNAs targeting CLUSTERIN(CLU), GRP78 and BAX and then exposed for 2 h to plasma from ITP patients and healthy controls. We investigated the expression of specific regulatory genes at mRNA level by qRT-PCR and at protein level by Western blot and immunofluorescence. We performed transmission electron microscopy (TEM) on primary MKs derived from PBMCs of ITP patients and controls. Data was statistically analysed by GraphPad Prism using One-way or Brown-Forsythe and Weltch ANOVA followed by multiple comparisons tests to compare the means between groups.

Results: We observed a significant upregulation of CLU,GRP78,GRP94,CASPASE-3,-8 and BAX in MEG-01 cells treated with ITP plasma compared to healthy plasma controls (p < 0.05). The increased mRNA expression of these markers could be reversed upon treatment with either Z-VAD-FMK or Rapamycin. mRNA levels of all investigated markers were significantly increased upon ABT737 and ITP plasma treatment compared to healthy controls. We further observed a significant increase of the expression of the autophagy (ATG5,LC3-II,LAMP-1) and ER-phagy markers (FAM134B,Atlastin-2) at mRNA level in ITP plasma versus healthy controls and/or Rapamycin treated cells. By TEM, ITP derived MKs showed a marked vacuolization of the cytoplasm, nuclear condensation and fragmentation in comparison to healthy MKs.

Conclusion(s): Our results revealed a significant impairment in the apoptosis and autophagy/ER phagy pathways in ITP indicating that these underlying mechanisms could be involved in the disease pathophysiology. These findings could be further used to identify promising molecular targets with therapeutic potential.