Platelets : present insights and future prospectives

number: 
805
إنجليزية
department: 
Degree: 
Imprint: 
Medicine
Author: 
Lola Leon Baghdasar Momjian
Supervisor: 
Dr.Faik Hussain Mohammed
Dr. Marwan S.M. Al-Nimer
year: 
2003
Abstract:

Platelets were tested for the production of nitric oxide and one of the reactive nitrogen oxide species; peroxynitrite. In addition, the effect of platelets on the endothelial cells was studied in terms of nitric oxide and peroxynitrite production, as well as, endothelial cells' DNA modification. Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared from blood samples obtained from apparently healthy adult volunteers who had not taken any medication for at least two weeks. Endothelial cells were extracted from the umbilical vein of human umbilical cords obtained from normal vaginal deliveries. The cells were isolated from the umbilical vein by infusion of the interior of the vessel with a mixture of enzymes (trypsin and pancreatin) with EDTA in normal saline. The isolated endothelial cells, alone or after incubation with PPP or PRP were used for nitric oxide, peroxynitrite, and DNA assays. In biological samples, nitric oxide was determined indirectly, by measuring the level of nitrate. And peroxynitrite was determined by measuring the level of nitrophenol which resulted from peroxynitrite-mediated nitration of phenol. No significant difference was found in nitric oxide level between PPP and PRP; however, peroxynitrite level was significantly higher in PRP compared to that of PPP. In addition, PRP incubated with epinephrine showed no significant change in nitric oxide or peroxynitrite level compared to that of PRP alone. When human umbilical vein endothelial cells (HUVECs) were incubated with either PPP or PRP (with or without epinephrine), there was a significant reduction in nitric oxide level compared to that of PPP or PRP alone, respectively. Moreover, there was a significant reduction in nitric oxide level in endothelial cells after incubation with PPP or PRP (with or without epinephrine) compared to the values obtained from the mathematical addition of that of the non-incubated endothelial cells plus that of PPP or PRP, respectively. However, :no significant difference was detected in nitric oxide level between HUVECs incubated with PPP and those incubated with PRP. On the other hand, a significant increase in peroxynitrite level was found in HUVECs incubated with PRP compared to those incubated with PPP. In addition, there was no significant difference in either nitric oxide or peroxynitrite level in endothelial cells incubated with PRP and those incubated with PRP already incubated with epinephrine. Taken together, these results suggested that platelets, whether activated or not, had interacted with the endothelial cells in vitro, and had a role in the production of peroxynitrite. And that the formation of peroxynitrite, from the reaction of nitric oxide and superoxide, presumably had masked the greater nitric oxide release resulting from the interaction of the platelets and the endothelial cells. Further experiments were conducted in this study to show the effect of platelets on the endothelial cells' DNA, in terms of separation of the DNA strands, measured as increase in the absorbance of DNA at 260 nm. A significant increase, was observed in the absorbance of DNA extracted from endothelial cells incubated with PRP, already incubated with epinephrine, in comparison to that of the non-incubated endothelial cells or the endothelial cells incubated with PRP without epinephrine. It was also shown that exogenously prepared peroxynitrite could cause strand separation of the DNA extracted from the endothelial cells. Therefore, it was suggested that activated platelets might have a harmful effect on DNA, at least in part, through peroxynitrite formation. In conclusion, platelets do not seem to contribute significantly to the plasma level of nitric oxide, but can be considered as a source of peroxynitrite in the plasma. Exposure of endothelial cells, in vitro, to resting or activated platelets, can result in increased production of peroxynitrite. This exposure can also cause DNA damage in the form erf separation of the endothelial cells' DNA strands, but onty if the platelets were activated.