Platelet free cytosolic calcium concentration during ageing of type 2 diabetic patients

The aim of this study was to examine the effects of biological ageing on concentration of free calcium ions ([Ca2+](i)) in platelets of type 2 diabetic patients, an issue not documented till now. Since diabetes platelets are surrounded by a hyperglycemic environment enriched in products of nonenzymatic glycation and oxidation of proteins (known as "advanced glycation end products'' (AGEs)), we questioned on the individual effect of high glucose concentration, AGEs, and oxidative stress on platelet [Ca2+](i). To these purposes, we performed: (i) measurement of basal and thrombin-stimulated [Ca2+](i) in platelets isolated from type 2 diabetic patients and from normal subjects of young (27 +/- 7 years), mature (48 +/- 12 years) and older (460 years) age, and (ii) quantitation of [Ca2+](i) when platelets of young healthy subjects were exposed to 25.5mM glucose (vs. 11mM glucose), 0.23-1.7mM AGE-poly-L-lysine (vs.poly-L-lysine),0.3-2.26 mM AGE-albumin (vs. albumin), and to 10mM and 100mM H2O2. Reactions of nonenzymatic glycation were conducted in vitro to prepare AGE-poly-L-lysine and AGE-albumin, and spectrofluorimetry was used to measure platelet free [Ca2+](i) following Fura-2/AM cells loading. The results showed that: (i) in physiological conditions, ([Ca2+](i)) was similar to 10% increased in the platelets of the mature subjects, and similar to 33% enhanced at the older group (vs. young), sustaining that biological ageing is associated with accumulation of free [Ca2+](i) within the platelets cytoplasm; (ii) in type 2 diabetes, [Ca2+](i) was similar to 16% and similar to 27% higher in the platelets of mature and older patients, respectively (vs. age-matched normals), demonstrating that ageing of diabetics is accompanied by alterations in calcium balance (vs. physiological ageing); (iii) thrombin (1U/ml) induced similar to 39% increase of [Ca2+](i) in platelets of matures and similar to 29% at older normals, and similar to 34% increase at the mature diabetics, similar to 84% at the older diabetics (vs. no thrombin condition), indicating that under thrombin stimulation simultaneous insults of diabetes and advanced age produced a higher thrombin-evoked mobilization of Ca2+ from intracellular stores; (iv) the components of the diabetic milieu had various effects on platelet free [Ca2+] i: high enhancement (similar to 73%) in 25.5mM glucose (vs. 11mM glucose), a minor increase (similar to 15%) in 100mM H2O2, and a decrease ( by similar to 56% and similar to 132%) in 1.7mM AGE-poly-L-lysine ( vs. poly-L-lysine) and 2.26mM AGE-albumin ( vs. albumin), respectively. Thus, a complex of factors contribute to platelet free [Ca2+](i) during biological ageing of diabetics: age, hyperglycemia and oxidative stress release Ca2+ from intracellular stores, while AGE products reduced the cytosolic free Ca2+. Thus, platelets [Ca2+](i) level is the final result of the combined effects of ageing and of the components of the diabetic milieu.