Department of Geriatrics, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Int J Vitam Nutr Res. 2010 Jun;80(3):168-77. doi: 10.1024/0300-9831/a000019.
Advanced glycation end products (AGEs) increase platelet aggregation and suppress vascular nitric oxide (NO) synthase (NOS) activity, and these effects may contribute to the atherothrombotic disease seen in diabetes. The aims of this study were to determine in vitro whether pyridoxine can abrogate the impairment in platelet NOS activity caused by AGEs, and to determine the mechanism by which it does this. Platelet aggregation was measured by Born aggregometry. Intraplatelet cyclic guanosine-3',5'-monophosphate (cGMP, an index of bioactive NO) was measured by radioimmunoassay. Serine-1177-specific phosphorylation of NOS type 3 (NOS-3) and phosphorylation of protein kinase Akt were determined in platelets by Western blotting. Phosphatidylinositol 3-kinase (PI3K) activity in platelets was ascertained by homogeneous time-resolved fluorescence (HTRF) assay. We found that AGE-modified albumin (AGEs) 200 mg/L increased platelet aggregability and decreased intraplatelet cGMP; these effects were largely attenuated by pyridoxine. Western blotting studies revealed that AGEs decreased NOS-3 phosphorylation on serine-1177, increased NOS-3 O-glycosylation, and decreased serine phosphorylation of protein kinase Akt; all of these changes were abrogated by pyridoxine. Direct measurement of PI3K activity in platelets demonstrated that all of the above effects could be attributed to a suppression by AGEs of PI3K activity, which was prevented by co-incubation with pyridoxine. We conclude that pyridoxine is effective in ameliorating the dysfunction of platelet NO signaling in response to AGEs, through improving PI3K activity, and hence downstream Akt phosphorylation and in turn serine-1177 phosphorylation of NOS-3.
糖基化终产物（AGEs）可增加血小板聚集并抑制血管一氧化氮合酶（NOS）活性，这些作用可能导致糖尿病患者发生动脉粥样血栓疾病。本研究旨在体外确定吡哆醇是否可以消除 AGE 引起的血小板 NOS 活性损伤，并确定其作用机制。采用 Born 聚集法测定血小板聚集。通过放射免疫法测定血小板内环鸟苷酸-3'，5'-单磷酸（cGMP，生物活性 NO 的指标）。通过 Western blot 测定血小板中 NOS 型 3（NOS-3）的丝氨酸-1177 特异性磷酸化和蛋白激酶 Akt 的磷酸化。通过均相时间分辨荧光（HTRF）测定法测定血小板中磷脂酰肌醇 3-激酶（PI3K）活性。我们发现 200mg/L 的 AGE 修饰白蛋白（AGEs）可增加血小板聚集性并降低血小板内 cGMP；这些作用在很大程度上可被吡哆醇减弱。Western blot 研究显示，AGEs 降低了 NOS-3 丝氨酸-1177 的磷酸化，增加了 NOS-3 的 O-糖基化，并降低了蛋白激酶 Akt 的丝氨酸磷酸化；吡哆醇可消除所有这些变化。直接测定血小板中 PI3K 活性表明，上述所有作用都可以归因于 AGE 对 PI3K 活性的抑制，吡哆醇共同孵育可防止这种抑制。我们的结论是，吡哆醇通过改善 PI3K 活性以及随后 Akt 磷酸化和 NOS-3 丝氨酸-1177 磷酸化，有效改善了血小板对 AGE 反应的一氧化氮信号转导功能障碍。
