Pyridoxine Improves Platelet Nitric Oxide Synthase Dysfunction In Response To Advanced Glycation End Products

Non-insulin dependent diabetes mellitus is one of the highestmorbidity and mortality of the disease worldwidely. Cardiovascularcomplications are the most frequent cause in diabetic patients. Thesecomplications include microangiopathy, retinopathy, neuropathy,nephropathy, and macroangiopathy, which can accelerate atherosclerosis.Considering the high frequency of cardiovascular diseases in diabetes, itis important to understand the diabetes pathogenesis. One postulatedhypothesis for the pathogenesis is abnormal platelet activation thatcontributes to both diabetic micro- and macroangiopathy. There isevidence supporting it. It's well known that platelets play an importantrole in the pathogenesis of atherosclerosis. And furthermore, it has beendemonstrated that platelet function in DM patients is enhanced. Avariety of mechanism may be responsible for enhanced plateletaggregation. Among them, hyperglycemia may represent a factor forplatelet activation. As we know that chronic hyperglycemia is a majorinitiator of vascular complications of diabetes. Varioushyperglycemia-induced metabolic and hemodynamic derangements,including increased advanced glycation end product (AGE) formation.It's reported that AGEs can directly increase the platelet aggregation and that's possibly with superoxide anions. However, the mechanismwhereby AGEs potentiate platelet aggregation is incompletelyunderstood. It has shown that AGEs impair the activity of endothelialnitric oxide (eNOS), which leading to endothelium dysfunction, onendothelium cell. While on platelets, it was still unclear.Since the identification of nitric oxide (NO) as the primaryendothelium-derived relaxing factor, an increasing body of evidence hasaccumulated to show that NO is more than a vasodilator as it exertsanti-platelet actions and plays a crucial role in the regulation of vascularhomeostasis. NO is synthesized from the amino acid L-arginine by afamily of enzymes called the NO synthases (NOS). Among of the threeisoforms (eNOS, iNOS and nNOS), it has reported that the isoformpresent in platelet is eNOS. How to regulate the bioactivity of eNOS isa new target in various cardiovascular diseases. While, much less isknown about the mechanisms determining its activity in platelets. Thereis a classical signaling transduction that the serine/threonine proteinkinase Akt/PKB mediates the activation of eNOS, leading to increasingNO production.Pyridoxine, a major component of vitamin B6, plays vital roles innumerous metabolic processes in the human body. Nowadays, it hasbeen associated with some benefits in non-randomised studies, such aslower blood homocysteine concentration, reduced platelet aggregation. And the feasible mechanism may be attributable to the role ofpyridoxine as a cofactor in several enzymatic reactions inatherosclerosis. Recent studies of our group also suggest a protectionagainst dysfunction of endothelial cell nitric oxide production inresponse to low-density lipoprotein, but the mechanism on platelets isstill unclear.The aims of the present study were firstly to determine whetherpyridoxine can abrogate the impairment in platelet NOS activity causedby AGEs. Serine-1177-specific phosphorylation, O-glycosylation ofNOS-3, and phosphorylation of protein kinase Akt were determined inplatelets by western blotting, and intracellular cGMP (an index ofbioactive NO) was measured by radioimmunoassay. Firstly, we detecteda marked concentration-dependent effect of pyridoxine on the plateletaggregation. And pyridoxine 2 mmol/l could attenuate the plateletaggregation induced by AGEs 200 mg/l. And secondly, we found thatpyridoxine could prevent the AGEs-induced changes of NOS-3 state inplatelets. AGEs decreased NOS-3 phosphorylation, and at the same timeincreased NOS-3 glycation. All of the above changes could beprevented by pyridoxine. While the total NOS haven't been changed. Atthe same time, cGMP had the corresponding changes. It suggested thatpyridoxine could ameliorate the dysfunction of blood platelet NOS inresponse to AGEs. Furthermore, AGE decreased the amount of phospho-Akt, with no change in total Akt, and this was also preventedby pyridoxine. And Ly294002 (the inhibitor of PI3k) can eliminate theeffection of pyridoxine on phosphorylation of eNOS and Akt. Theresults of PI3k activation suggested that pyridoxine can increase theactivation of PI3k, while AGEs decrease. And pyridoxine can reversethe effection of AGEs. The initially conclusion was that pyridoxineameliorates the dysfunction of platelet NOS in response to AGE,through PI3k-Akt-eNOS-cGMP. This has important potentialtherapeutic implications for the use of pyridoxine in the preventionand/or treatment of diabetes-related atherosclerosis.