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 ([Ca(2+)](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 [Ca(2+)](i). To these purposes, we performed: (i) measurement of basal and thrombin-stimulated [Ca(2+)](i) in platelets isolated from type 2 diabetic patients and from normal subjects of young (27 +/- 7 years), mature (48 +/- 12 years) and older (>60 years) age, and (ii) quantitation of [Ca(2+)](i) when platelets of young healthy subjects were exposed to 25.5 mM glucose (vs. 11 mM glucose), 0.23-1.7 mM AGE-poly-L-lysine (vs. poly-L-lysine), 0.3-2.26 mM AGE-albumin (vs. albumin), and to 10 mM and 100 mM H(2)O(2). 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 [Ca(2+)](i) following Fura-2/AM cells loading. The results showed that: (i) in physiological conditions, [Ca(2+)](i) was approximately 10% increased in the platelets of the mature subjects, and approximately 33% enhanced at the older group (vs. young), sustaining that biological ageing is associated with accumulation of free [Ca(2+)](i) within the platelets cytoplasm; (ii) in type 2 diabetes, [Ca(2+)](i) was approximately 16% and approximately 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 approximately 39% increase of [Ca(2+)](i) in platelets of matures and approximately 29% at older normals, and approximately 34% increase at the mature diabetics, approximately 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 Ca(2+) from intracellular stores; (iv) the components of the diabetic milieu had various effects on platelet free [Ca(2+)](i): high enhancement ( approximately 73%) in 25.5 mM glucose (vs. 11 mM glucose), a minor increase ( approximately 15%) in 100 mM H(2)O(2), and a decrease (by approximately 56% and approximately 132%) in 1.7 mM AGE- poly-L-lysine (vs. poly-L-lysine) and 2.26 mM AGE- albumin (vs. albumin), respectively. Thus, a complex of factors contribute to platelet free [Ca(2)(+)](i) during biological ageing of diabetics: age, hyperglycemia and oxidative stress release Ca(2)(+) from intracellular stores, while AGE products reduced the cytosolic free Ca(2+). Thus, platelets [Ca(2)(+)](i) level is the final result of the combined effects of ageing and of the components of the diabetic milieu.