title: Pyridoxine improves platelet nitric oxide synthase dysfunction induced by advanced glycation end products in vitro.
abstract: 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.
authors.affiliation: Department of Geriatrics, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Han
authors.forename: Yuan
authors.initials: Y
authors.lastname: Liu
authors.forename: Qiongyu
authors.initials: Q
authors.lastname: Mi
authors.forename: Liping
authors.initials: L
authors.lastname: Xie
authors.forename: Yan
authors.initials: Y
authors.lastname: Huang
authors.forename: Qin
authors.initials: Q
authors.lastname: Jiang
authors.forename: Qi
authors.initials: Q
authors.lastname: Chen
authors.forename: Albert
authors.initials: A
authors.lastname: Ferro
authors.forename: Naifeng
authors.initials: N
authors.lastname: Liu
authors.forename: Yong
authors.initials: Y
authors.lastname: Ji
mesh_terms: D000328:Adult; D001792:Blood Platelets / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D003925:Diabetic Angiopathies / Q000517:prevention & control; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D017127:Glycation End Products, Advanced / Q000037:antagonists & inhibitors* / Q000494:pharmacology / Q000502:physiology; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D008297:Male; D009569:Nitric Oxide / Q000096:biosynthesis*; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011736:Pyridoxine / Q000494:pharmacology*; D012709:Serum Albumin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D000075462:Serum Albumin, Human; D015398:Signal Transduction / Q000187:drug effects; D055815:Young Adult
chemical_list: D004791:Enzyme Inhibitors; D017127:Glycation End Products, Advanced; D010975:Platelet Aggregation Inhibitors; D012709:Serum Albumin; C109748:advanced glycation end products-human serum albumin; D009569:Nitric Oxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D006152:Cyclic GMP; D011736:Pyridoxine; D000075462:Serum Albumin, Human