OP3 Identification and examination of Mitsugumin 23 within platelets

Intracellular zinc and calcium dynamics are important for both platelet activation and function. We have recently shown that the Ca²⁺ permeable cation channel mitsugumin 23 (MG23), found in the endoplasmic/sarcoplasmic reticulum of other cell types, is Zn²⁺-sensitive and may facilitate crosstalk between the two metal ions. The role of MG23 in platelet responses has never been explored but given its suggested role as a Ca²⁺ leak channel we suggest that MG23 may contribute to the elevation of intracellular Ca²⁺ resulting in hyperactive platelets. The aim of this study was to confirm the presence of MG23 in platelets, identify its subcellular localization and evaluate the role of MG23 in platelet aggregation by comparing the aggregatory responses of platelets prepared from wild type and MG23-KO mice. Expression of MG23 was assessed using RT-PCR and western blotting using mRNA and protein extracted from healthy donor platelets and megakaryocyte-like cells (MEG-01 cell line). Platelet aggregation assays were carried out on washed platelets from both WT and MG23-KO mice to evaluate the effect of MG23 knockout on platelet clotting. The institutional ethics committee at the University of St. Andrews approved the study. Work was carried out under project licence P82006EDF. Subcellular localization of MG23 within platelets was assessed by confocal microscopy using fixed MEG-01 cells. MG23 mRNA and protein were identified in both platelets and MEG-01 cells. The staining of platelets with antibodies directed against SERCA2 ATPase and MG23 yielded colocalized fluorescence, indicative of MG23 localization to the dense tubular system. Both WT and MG23-KO murine platelets aggregated in response to various platelet agonists. MG23 expression was confirmed within platelets and is present in the dense tubular system. Though it has been proposed to serve as a Ca²⁺ leak channel in other cell types, its specific role in platelets is less clear.