Abstract P2084: Determining The Role Of Rap1b In Megakaryocyte Survival

Abstract

Platelet counts are tightly regulated through production by megakaryocytes and clearance by hepatocytes. Thrombotic risk and poor cancer prognosis correlate with platelet counts on the high and low ends of the normal range, respectively. Therefore, understanding the factors that regulate megakaryocyte survival and function are important for human health. The small GTPase, Rap1b increases in abundance as megakaryocytes mature and mediates survival in other cell types by preventing apoptosis. We hypothesize that disruption of the Rap1b gene in the megakaryocytic leukemia cell line, DAMI, will limit cell survival by increasing susceptibility to apoptosis. We used CRISPR/Cas to specifically disrupt the Rap1b gene, resulting in no detectable Rap1b protein in DAMI cells. Compared to wild-type DAMI cells, Rap1b knockout cells had nearly a 50% reduction in survival as measured by metabolic activity using an MTT-assay. To assess apoptosis susceptibility, we monitored mitochondrial membrane potential using the fluorescent sensor JC-1 and caspases activity using the Fluorochrome Inhibitor of Caspases (FLICA). These results suggest that Rap1b promotes megakaryocyte survival.