Alexandru Nicoleta, Popov Doina, Sbarcea Adriana, Amuzescu Manuela
Institute of Cellular Biology and Pathology Nicolae Simionescu, Bucharest, Romania.
Platelets. 2007 Nov;18(7):473-80. doi: 10.1080/09537100701507619.
The aim of this study was to examine the effects of biological ageing on concentration of free calcium ions (Ca(2+)) 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+). To these purposes, we performed: (i) measurement of basal and thrombin-stimulated Ca(2+) 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+) 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+) following Fura-2/AM cells loading. The results showed that: (i) in physiological conditions, Ca(2+) 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+) within the platelets cytoplasm; (ii) in type 2 diabetes, Ca(2+) 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+) 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+): 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)(+) 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)(+) level is the final result of the combined effects of ageing and of the components of the diabetic milieu.
本研究旨在探讨生物衰老对2型糖尿病患者血小板中游离钙离子（Ca(2+)）浓度的影响，这一问题迄今尚无文献记载。由于糖尿病患者的血小板处于富含蛋白质非酶糖基化和氧化产物（即“晚期糖基化终产物”（AGEs））的高血糖环境中，我们对高糖浓度、AGEs和氧化应激对血小板Ca(2+)的个体影响提出疑问。为此，我们进行了以下操作：（i）测量从2型糖尿病患者以及年轻（27±7岁）、成熟（48±12岁）和老年（>60岁）正常受试者分离的血小板中基础和凝血酶刺激后的Ca(2+)；（ii）当年轻健康受试者的血小板暴露于25.5 mM葡萄糖（对比11 mM葡萄糖）、0.23 - 1.7 mM AGE-聚-L-赖氨酸（对比聚-L-赖氨酸）、0.3 - 2.26 mM AGE-白蛋白（对比白蛋白）以及10 mM和100 mM H(2)O(2)时，对Ca(2+)进行定量。通过体外进行非酶糖基化反应制备AGE-聚-L-赖氨酸和AGE-白蛋白，并在Fura-2/AM细胞加载后使用荧光分光光度法测量血小板游离Ca(2+)。结果显示：（i）在生理条件下，成熟受试者血小板中的Ca(2+)大约增加10%，老年组（对比年轻组）大约增加33%，表明生物衰老与血小板细胞质内游离Ca(2+)的积累有关；（ii）在2型糖尿病患者中，成熟和老年患者血小板中的Ca(2+)分别比年龄匹配的正常受试者高约16%和约27%，表明糖尿病患者的衰老伴随着钙平衡的改变（对比生理衰老）；（iii）凝血酶（1U/ml）使成熟正常受试者血小板中的Ca(2+)增加约39%，老年正常受试者增加约29%，成熟糖尿病患者增加约34%，老年糖尿病患者增加约84%（对比无凝血酶条件），表明在凝血酶刺激下，糖尿病和高龄的同时损伤导致细胞内钙库中凝血酶诱发的Ca(2+)动员更高；（iv）糖尿病环境的成分对血小板游离Ca(2+)有不同影响：25.5 mM葡萄糖（对比11 mM葡萄糖）时显著增加（约73%），100 mM H(2)O(2)时轻微增加（约15%），1.7 mM AGE-聚-L-赖氨酸（对比聚-L-赖氨酸）和2.26 mM AGE-白蛋白（对比白蛋白）时分别降低（约56%和约132%）。因此，在糖尿病患者的生物衰老过程中，多种因素共同影响血小板游离Ca(2+)：年龄、高血糖和氧化应激从细胞内钙库释放Ca(2+)，而AGE产物降低细胞质游离Ca(2+)。因此，血小板Ca(2+)水平是衰老和糖尿病环境成分综合作用的最终结果。
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