Pyridoxine Increases Nitric Oxide Biosynthesis In Human Platelets

Atherosclerosis(AS) is a kind of damaged endothelial cells, smooth muscle cells lining blood vessel invasion, vascular smooth muscle cell proliferation mainly of the pathological process of vascular remodelling,and its incidence is complex and exact cause has not been fully clarified. Numerous studies confirm that platelet activation and aggregation in the occurrence and development of AS has played an important role.Nitric oxide (NO), the primary endothelium-derived relaxing factor, participates in variety of biological activities in vivo, such as vasorelaxation , inhibition of vascular smooth muscle cell proliferation, and inhibition of vascular permeability.At the same time a growing number of studies confirmed the role of NO addition to the above, but also can inhibit platelet aggregation to maintain a balanced environment within the blood vessels, thus preventing the development of cardiovascular diseases. NO is synthesized from the amino acid L-arginine by a family of enzymes called the NO synthases(NOS). Among of the three isoforms(eNOS, iNOS, nNOS), it is reported that the isoform present in platelet is eNOS. While much less is known about the mechanisms determining its activity in platelet.There is a classical signaling transduction that the serine/threonine protein kinase Akt/PKB mediates the activation of eNOS, leading to increasing NO production.Pyridoxine, a major component of vitamin B6, palys vital roles in numerous metabolic processes in the human body. Nowadays, it has been associated with some benefits in non-randomised studies, such as lowing blood homocyserine concentration, reducing platelet aggregation. And the feasible mechanism may be attributable to the role of pyridoxine as a cofactor in several enzymatic reactions in atherosclerosis. But the mechanism on platelets is still unclear.The purpose of this study was firstly to determine whether pydidoxine can increase the platelet NO synthesis in vitro, followed by studing its mechanism of this role. The platelets were extracted from the healthy human to observe whether pydidoxine can inhibit their aggregation induced by ADP or Thrombin,and cGMP(an index of bioactive NO) was measured by radioimmunoassay. eNOS Ser-1177-specific phosphorylation and phosphorylation of protein kinase Akt were determined in platelets by western blot .The activity of phosphatidylinositol-3-kinase was analysed by HTRF.Firstly, we detected a marked concentration-dependent effect of pyridoxine on the platelet aggregation induced by ADP or Thrombin. When the concentration of pyridoxine was 2mmol/L, its ability to inhibit platelet aggregation is no longer increased as the concentration increases. RIA results showed that pyridoxine can increase the biological activity of NO. We further found that , pyridoxine can increase eNOS Ser-1177 phosphorylation and Akt serine phosphorylation, it can also increase PI3K activity and this effect can be inhibited by PI3K inhibitor LY294002. The results of this study show that, pyridoxine can effectively inhibit the ADP or Thrombin-induced platelet activation and increased the biosynthesis of NO on platelet. Pyridoxine play this role by improving the activity of PI3K, thereby increasing the downstream Akt phosphorylation, further increase in eNOS Ser-1177 phosphorylation site. This has important potential therapeutic implications for the use of pyridoxine in the prevention and/or treatment of Thrombosis-related disease.