Chair of Clinical Biochemistry, Department of Laboratory Medicine, Medical University of Gdańsk, Dębinki 7, 80-211, Gdańsk, Poland.
Chair of Clinical Biochemistry, Department of Molecular Medicine, Medical University of Gdańsk, Gdańsk, Poland.
Pharmacol Rep. 2020 Feb;72(1):225-237. doi: 10.1007/s43440-019-00005-0. Epub 2020 Jan 10.
Hyperactivation of blood platelets is an essential factor in the pathomechanism of diabetes-evoked angiopathies. The aim of this work was to investigate whether blood platelets hyperactivation resulting from type 2 diabetic hyperglycaemia-increased pyruvate dehydrogenase complex activity and excessive acetyl-CoA accumulation may be brought to the normal range by the enzyme inhibitors.
Platelets were isolated from the blood of 9 type 2 diabetic patients and 10 healthy donors. Effects of 3-bromopyruvate and 3-nitropropionate on pyruvate dehydrogenase complex (PDHC) and succinate dehydrogenase activities, as well as levels of acetyl-CoA, ATP, thiobarbituric acid reactive species and aggregation were assessed in non-activated and thrombin-activated platelets.
In type 2 diabetic patients fasting plasma glucose and fructosamine levels were 61 and 64% higher, respectively, than in the healthy group (p < 0.001). In non-activated diabetic platelets PDHC activity, PDHC-E2, acetyl-CoA and ATP levels were 66, 70, 68 and 60%, higher, respectively, than in platelets from healthy controls (p < 0.01). 3-bromopyruvate (0.1 mM) decreased pyruvate dehydrogenase activity in healthy and diabetic platelets by 42% and 59%, respectively. Similar inhibitory effects were observed for acetyl-CoA and ATP levels, aggregation and TBARS accumulation rates. Succinate dehydrogenase activity was inhibited by 3-nitropropionate (10 mM) to 38 and 41% of control values in healthy and diabetic platelets, respectively, but affected neither function nor acetyl-CoA metabolism in platelets of both groups.
These data indicate that inhibition of pyruvate dehydrogenase excessive activity in diabetic platelets by 3-bromopyruvate may normalise their functional parameters through adjustment of acetyl-CoA/ATP levels to control values. Platelets from blood of diabetic patients display higher activities of pyruvate dehydrogenase complex (PDHC), higher levels of dihydrolipoate transacetylase (DLAT, E2 subunit of PDHC) as well as higher levels of acetyl-CoA yielding greater ATP/ADP accumulation than in platelets of normoglycemic subjects. Therefore, in diabetic platelets, thrombin caused higher release of ATP/ADP triggering excessive production of reactive oxygen species (ROS) and stronger aggregation compared to control platelets. In diabetic platelets, relative excess of DLAT in PDHC made them highly susceptible to 3-bromopyruvate (3BrP) inhibition. Resulting limitation of acetyl-CoA provision by 3-BrP normalised activity of diabetic platelets.
血小板的过度激活是糖尿病引发血管病变的病理机制中的一个重要因素。本研究旨在探讨 2 型糖尿病患者由于高血糖导致丙酮酸脱氢酶复合体（PDHC）活性增加和乙酰辅酶 A 过度积累而引起的血小板过度激活，是否可以通过酶抑制剂恢复到正常水平。
从 9 名 2 型糖尿病患者和 10 名健康供体的血液中分离血小板。在非激活和凝血酶激活的血小板中评估 3-溴丙酮酸和 3-硝基丙酸对 PDHC 及琥珀酸脱氢酶活性的影响，以及乙酰辅酶 A、ATP、硫代巴比妥酸反应性物质和聚集的水平。
2 型糖尿病患者的空腹血糖和果糖胺水平分别比健康组高 61%和 64%（p<0.001）。在非激活的糖尿病血小板中，PDHC-E2、乙酰辅酶 A 和 ATP 水平分别比健康对照组高 66%、70%、68%和 60%（p<0.01）。0.1 mM 的 3-溴丙酮酸使健康和糖尿病血小板中的丙酮酸脱氢酶活性分别降低 42%和 59%。对乙酰辅酶 A 和 ATP 水平、聚集和 TBARS 积累率也观察到类似的抑制作用。10 mM 的 3-硝基丙酸使健康和糖尿病血小板中的琥珀酸脱氢酶活性分别抑制至对照值的 38%和 41%，但对两组血小板的功能和乙酰辅酶 A 代谢均无影响。
这些数据表明，通过 3-溴丙酮酸抑制糖尿病血小板中丙酮酸脱氢酶的过度活性，可以通过将乙酰辅酶 A/ATP 水平调节至对照值来使血小板的功能参数正常化。来自糖尿病患者血液的血小板表现出更高的丙酮酸脱氢酶复合体（PDHC）活性、更高的二氢乳清酸转乙酰酶（DLAT，PDHC 的 E2 亚基）水平以及更高的乙酰辅酶 A 水平，从而导致与正常血糖个体相比更大的 ATP/ADP 积累。因此，与对照血小板相比，凝血酶引起糖尿病血小板中更高的 ATP/ADP 释放，引发过量的活性氧（ROS）产生和更强的聚集。在糖尿病血小板中，PDHC 中的 DLAT 相对过剩使它们极易受到 3-溴丙酮酸（3BrP）的抑制。由此导致的 3-BrP 对乙酰辅酶 A 供应的限制使糖尿病血小板的活性正常化。
