Altered platelet calsequestrin abundance, Na+/Ca2+ exchange and Ca2+ signaling responses with the progression of diabetes mellitus

Introduction: Downregulation of calsequestrin (CSQ), a major Ca2+ storage protein, may contribute significantly to the hyperactivity of internal Ca2+ ([Ca2+](i)) in diabetic platelets. Here, we investigated changes in CSQ-1 abundance, Ca2+ signaling and aggregation responses to stimulation with the progression of diabetes, especially the mechanism(s) underlying the exaggerated Ca2+ influx in diabetic platelets. Materials and methods: Type 1 diabetes was induced by streptozotocin in rats. Platelet [Ca2+](i) and aggregation responses upon ADP stimulation were assessed by fluorescence spectrophotometry and aggregometry, respectively. CSQ-1 expression was evaluated using western blotting. Results: During the 12-week course of diabetes, the abundance of CSQ-1, basal [Ca2+](i) and ADP-induced Ca2+ release were progressively altered in diabetic platelets, while the elevated Ca2+ influx and platelet aggregation were not correlated with diabetes development. 2-Aminoethoxydiphenyl borate, the store-operated Ca2+ channel blocker, almost completely abolished ADP-induced Ca2+ influx in normal and diabetic platelets, whereas nifedipine, an inhibitor of the nicotinic acid adenine dinucleotide phosphate receptor, showed no effect. Additionally, inhibition of Na+/Ca2+ exchange induced much slower Ca2+ extrusion and more Ca2+ influx in normal platelets than in diabetic platelets. Furthermore, under the condition of Ca2+-ATPase inhibition, ionomycin caused greater Ca2+ mobilization and Ca2+ influx in diabetic platelets than in normal platelets. Conclusions: These data demonstrate that platelet hyperactivity in diabetes is caused by several integrated factors. Besides the downregulation of CSQ-1 that mainly disrupts basal Ca2+ homeostasis, insufficient Na+/Ca2+ exchange also contributes, at least in part, to the hyperactive Ca2+ response to stimulation in diabetic platelets. (C) 2014 Elsevier Ltd. All rights reserved.