Study on the intracellular mechanism of ADP-induced platelet aggregation inhibited by pyridoxal-5-phosphate

• Main Authors: Hsin-yi, Hsieh, 謝欣宜
• Other Authors: Sue-Joan, Chang
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/38829307205991208042

碩士 === 國立成功大學 === 生物學系 === 89 === ADP-induced platelet aggregation plays an important role in homeostasis and thrombosis. Pyridoxal-5-phosphate (PLP), a biological active form of vitamin B6, has been shown to inhibit in vitro and in vivo platelet aggregation induced by ADP. However, the inhibitory mechanism by which PLP operates is still not well understood. ADP activates platelets through surface receptors resulting in shape change, aggregation, and release of granule contents e. g. ATP release. ADP also cause a number of intracellular events including mobilization of calcium from intracellular stores, rapid calcium influx and decreased of intracellular adenosine 3-5''-cyclic monophsphate (cAMP) levels. The purpose of this study was to elucidate the signaling of PLP on ADP-induced platelet aggregation in relation to shape change, aggregation, secretion (ATP release) and intracellular calcium [Ca]i2+ mobilization and cAMP level. ADP-induced platelet aggregation of washed human platelet treated with PLP (100, 200, 300, 500 or 600 nM) for different duration (10, 20, 30, 40 or 50 min) in the presence of 2 mg/ml fibrinogen and 1 mM calcium was determined by Pack-4 aggregometer (Beaumont, Texas). The optimal time for PLP treatment was determined by the minimal time of treatment reguired to cause significant aggregation with minimal IC50 which was defined as the PLP concentration resulting in 50% inhibition of aggregation. The result showed that 10 min was the optimal time for PLP treatment. Therefore, platelets were treated with PLP for 10 min for the rest of the experiments throughout the study. Washed human platelets loaded with Fura-2/AM and treated with different concentrations of PLP (100, 200, 300, 500 or 600 nM) for 10 min were used to monitor [Ca]i2+ mobilization by F-4500 spectrophotometer (Hitachi) in the presence or absence of 1mM calcium. The results showed that ADP-induced platelet [Ca]i2+ mobilization were decreased with increasing concentrations of PLP in both absence and presence of 1mM calcium. Futher experiment indicated that PLP inhibited intracellular [Ca]i2+ release instead of extracellular [Ca]i2+ influx. Intracellular cAMP level of platelet was analyzed in the platelet-rich plasma (PRP) treated with PLP (100, 200, 300, 500 or 600 nM) for 10 min by EIA kit. The results showd that intracellular cAMP level was increased with increasing concentrations of PLP. Intracellular ATP release of platelet was analyzed in the washed human platelet (WPS) treated with PLP (100, 200, 300, 500 or 600 nM) for 10 min by Lucifrease-luciferin reagent. The result showed that PLP 500 nM inhibited ADP-induced ATP-release in washed human platelet, indicating that hight concentration of PLP had inhibited ADP-induced ATP-release in platelet. Scanning electron microscopy (SEM) observation showed that platelet pretreated with PLP (200, 300, 500 or 600 nM) for 10 min had shape change which compared with platelet treated with PLP 0 and 100 nM. In conclusion, PLP may inhibit [Ca]i2+release, ATP release and increase in cAMP level. PLP may also inhibited platelet shape change induced by ADP. These results suggest that PLP may inhibit ADP-induced platelet activation through the inhibition of intracellular [Ca]i2+release, ATP release , shape change and increase in cAMP level.