The balance of life and death may hinge on the intracellular localization of the small GTPase, Rap1b in the megakaryocyte cell line (DAMI)

Abstract

Platelets are specialized cell fragments that circulate in the bloodstream and clot to prevent blood from leaving damaged vessels. The number of circulating platelets is governed by the balance of platelet production by mature megakaryocytes and clearance of platelets from the bloodstream. Normal platelet function requires the signaling protein, Rap1b, whose abundance also increases as megakaryocytes mature. The intracellular location of Rap1b is determine by its phosphorylation state. When Rap1b is not phosphorylated, it associates with membranes; conversely when Rap1b is phosphorylated, it detaches from membranes and reside in the cytosol. We hypothesize that Rap1b, specifically in its phosphorylated, cytosolic state, promotes megakaryocytes survival and proliferation. To address this hypothesis, the human megakaryocyte cell line (DAMI cells) have been modified 1) by disrupt the rap1b gene creating a Rap1b‐knockout cell line, 2) stable overexpression of a Rap1b phospho‐mimetic mutant, 3) stable overexpression of a Rap1b phospho‐deficient mutant or 4) stable overexpression of wild‐type Rap1b (to be used as a control). Using the modified DAMI cell lines we are beginning to understand the impact of Rap1b phosphorylation on proliferation on in MTS survival assays. Changes in cell survival can be attributed to changes in cell cycle progression and/or susceptibility to apoptosis. To address possible changes in cell cycle progression, we have evaluated each cell line based on the percentage of cell undergoing DNA synthesis at a given time by BrdU incorporation assays. To evaluate apoptosis susceptibility, mitochondrial membrane integrity and activation/cleavage of Caspase 9 was determined in the presence or absence of the apoptotic inducer ABT‐737. The analysis of the role of Rap1b in megakaryocytes has the potential to increase our collective understanding of regulation of platelet count and could be a novel target to combat thrombocytopenia (low platelet count) or thrombocythemia (high platelet count).Support or Funding InformationUniversity of Wisconsin ‐LaCrosse Faculty Research Grant