Abstract
Mutations in genes that play a role in the processes of megakaryopoiesis and platelet production cause thrombocytopenia. Five pathogenic variants in the gene encoding cytochrome c (CYCS) associated with mild autosomal dominant thrombocytopenia have been reported. The role of cytochrome c in megakaryopoiesis and platelet formation is unknown, and it is unclear how mutations in CYCS cause thrombocytopenia. In the bone marrow, haematopoietic stem cells undergo a complex differentiation process in response to thrombopoietin, ultimately maturing into megakaryocytes (MKs). These mature MKs release platelets into the bloodstream through elongated intravascular projections called proplatelets. Culturing peripheral blood CD34+ and CD45+ cells from the G42S variant showed an acceleration in megakaryopoiesis compared to wild type (WT).
Earlier studies have demonstrated increased caspase activity in human G42S and Y49H (reported variants in CYCS) compared to WT cytochrome c in vitro. However, caspase activity in peripheral blood mononuclear cells (PBMCs) isolated from peripheral blood of the G42S variant showed no significant differences compared to WT. The involvement of the intrinsic apoptosis pathway in platelet production remains a topic of ongoing debate. To gain further insights into the molecular mechanisms of CYCS-associated thrombocytopenia, the c.145T>C mutation (Y49H) was introduced into K-562 and WTC11 iPSC cells using CRISPR-Cas base-editing. Inducing apoptosis in WTC11 iPSC cells with H2O2 revealed no significant differences in caspase activity between homozygous WT and homozygous mutant cells. However, this should be repeated as there were some technical differences. Moreover, H2O2 and ABT-737 were unable to induce apoptosis in the K-562 cells.
A previous study demonstrated that both Y49H and G42S variants reduce mitochondrial respiration in mouse cells. Another study found no significant differences in respiration function between the G42S variant and WT cytochrome c, as indicated by the relative oxygen consumption in mitoplasts lacking endogenous cytochrome c and containing either WT or G42S cytochrome c. Notably, the result of this study showed the c.145T>C mutation did not affect mitochondrial respiration in K-562 cells.
In summary, the findings of this study provide additional insights into the cellular basis of the Y49H variant in megakaryopoiesis. The results support that the c.145T>C mutation enhances MK maturation in K-562 cells under both 18.5% and 5% O2. Additionally, the c.145T>C mutation does not impact mitochondrial respiration function in the K-562 cells and apoptosis in WTC11 iPSC cells. Furthermore, culturing CD45+ cells isolated from peripheral blood revealed that under 5% O2, unlike in 18.5% O2, the CD45+ cells did not differentiate into MKs.