Atherosclerosis Research Centre, Key Laboratory of Human Functional Genomics, and the Affiliated Ophthalmic Hospital of Nanjing Medical University, Nanjing 210029, China.
Int J Vitam Nutr Res. 2009 Mar;79(2):95-103. doi: 10.1024/0300-9831.79.2.95.
Activation and aggregation of platelets are key events in the pathophysiology of thrombotic diseases. There is increasing evidence that platelet-derived nitric oxide (NO) exerts important anti-platelet actions. Pyridoxine may have beneficial therapeutic effects in cardiovascular disease states, and has previously been shown to increase endothelial NO biosynthesis. The aims of the present study were firstly to determine in vitro whether pyridoxine can increase platelet NO synthesis, and secondly to investigate the mechanism by which it does this. Platelets isolated from blood taken from healthy subjects were treated with pyridoxine or vehicle. 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 NO synthase 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. Our results showed that pyridoxine largely inhibited the aggregation of platelets in response to adenosine diphosphate (ADP) or thrombin and increased bioactive NO. It also increased NOS-3 phosphorylation on serine-1177, and increased Akt serine phosphorylation. PI3K activity was augmented by pyridoxine, an effect inhibited by the specific PI3K antagonist LY294002. In conclusion, pyridoxine is effective in elevating platelet NO biosynthesis, through improving PI3K activity and hence downstream Akt phosphorylation, and in turn serine-1177 phosphorylation of NOS-3. These data reveal a novel mechanism by which NOS-3 activity can be regulated in platelets.
血小板的激活和聚集是血栓性疾病病理生理学中的关键事件。越来越多的证据表明，血小板衍生的一氧化氮（NO）发挥重要的抗血小板作用。吡哆醇在心血管疾病状态下可能具有有益的治疗作用，并且先前已经显示可增加内皮细胞 NO 的生物合成。本研究的目的首先是确定吡哆醇是否可以在体外增加血小板的 NO 合成，其次是研究其作用机制。用吡哆醇或载体处理从健康受试者血液中分离出的血小板。通过 Born 聚集测定法测量血小板聚集。通过放射免疫测定法测量血小板内环鸟苷酸-3'，5'-单磷酸（cGMP，生物活性 NO 的指标）。通过 Western blot 法测定血小板中 NO 合酶 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）测定法确定血小板中的磷脂酰肌醇 3-激酶（PI3K）活性。我们的结果表明，吡哆醇在很大程度上抑制了血小板对二磷酸腺苷（ADP）或凝血酶的反应性聚集，并增加了生物活性 NO。它还增加了 NOS-3 丝氨酸-1177 的磷酸化，并增加了 Akt 丝氨酸磷酸化。吡哆醇增强了 PI3K 活性，这一作用被特异性 PI3K 拮抗剂 LY294002 抑制。总之，吡哆醇通过改善 PI3K 活性和随后 Akt 磷酸化，从而丝氨酸-1177 磷酸化 NOS-3，有效地提高血小板中 NO 的生物合成。这些数据揭示了一种调节血小板中 NOS-3 活性的新机制。
