3-bromopyruvate affects blood platelets responses in type 2 diabetes.
Hyperactivity of blood platelets is an essential factor in pathomechanism of diabetes-evoked angiopathies. The aim of this work was to investigate whether blood platelets hyperactivity 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 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, 64%, respectively higher 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%, respectively higher than in platelets from healthy controls (p<0.01). 3-bromopyruvate (0.1mM) 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 (10mM) 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 normalize their functional parameters though adjustment of acetyl-CoA/ATP levels to control values.
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Enzyme assays For enzyme assays collected post-incubation platelets were homogenized and solubilized in 0.2% v/v Triton X-100. The activities of hexokinase, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 22.214.171.124), 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate hydro-lyase(2-dehydro-3-deoxy-6-phospho-D-gluconate forming), EC 126.96.36.199), pyruvate dehydrogenase complex, aconitase (citrate(isocitrate)hydro lyase, EC 188.8.131.52), α-ketoglutarate dehydrogenase complex (2-oxoglutarate:lipoate oxidoreductase acceptor acylating, KDHC), succinate dehydrogenase (succinate:quinone oxidoreductase, EC 184.108.40.206) were assayed by spectrophotometric methods described elsewhere . Immediately before the assays, platelet membranes were solubilized by the addition of Triton X-100 at final concentration 0.2% by volume. Assays were performed at 37oC in Ultrospec 3 spectrophotometer (Amersham-LKB, Cambridge UK). 2.6 ATP/ADP Collected platelet pellet was deproteinized with 4% HClO4 and ATP/ADP contents were assessed by a luminometric method using luciferin/luciferase method on Berthold Junior LB 9509 luminometer, (Berthold Technology, Bad-Wildbad, Germany)]. Acetyl-CoA assay Whole platelet acetyl-CoA content and its subcellular compartmentalization was assayed, by the cycling method, as described earlier [8, 17]. Platelet pellet was deproteinized by addition of 0.08 mL of 5 mmol/L HCl and placement for 1 min in a boiling bath. For assessment mitochondrial acetyl-CoA levels platelet were solubilised in 20 mmol/L Tris-HCl buffer (pH 7.4), 125 mmol/L KCl, 3 mmol/L EDTA, and 1.4 g/L digitonin , layered over a 0.5-mL mixture of silicon oils (AR-20 and AR-200, 1:2 by volume) and mitochondria were collected by centrifugation through silicon oil layer and deproteinized as above . 2.8 Platelet aggregation and thiobarbituric acid reactive species (TBARS) assays Platelets were suspended in 0.3 ml of medium containing Tyrode’s buffer (pH 7.4) and 2.5 mmol/L glucose to obtain density of 200-300 x 103/mL and preincubated for 5 min at 37oC in APACT aggregometer (Labor, Ahrensburg, Germany), with simultaneous recording of spontaneous aggregation. The aggregation was activated by the addition of thrombin (0.1 U/mL) and continued for 10 min. Reaction was terminated with 10% trichloroacetic acid and accumulation of TBARS was assessed as described elsewhere .