PRT318对胶原诱导人血小板Syk磷酸化的影响

Effect of PRT318 on collagen-induced Syk phosphorylation in human platelets

目的观察脾酪氨酸激酶(Syk)抑制剂PRT060318(PRT318)在体外对人血小板聚集率及Syk磷酸化的影响。方法采用比浊法测定PRT318对胶原、二磷酸腺苷(ADP)及花生四烯酸(AA)诱导的人血小板聚集率的影响;运用Western blot检测Syk525/6磷酸化水平。结果 Syk特异性抑制剂PRT318可抑制胶原诱导的人血小板聚集率,但对ADP或AA诱导的人血小板聚集率无影响。在蛋白水平上PRT318可促进人血小板在胶原诱导下Syk525/6磷酸化水平升高。酪氨酸蛋白激酶抑制剂PP2可抑制胶原诱导的人血小板聚集,降低人血小板在胶原诱导下Syk525/6磷酸化水平,并可阻断胶原诱导下PRT318促Syk525/6磷酸化作用。结论 PRT318对胶原诱导的血小板聚集具有抑制作用,并能促进胶原诱导的血小板膜受体糖蛋白VI信号通路上Syk525/6磷酸化,提示PRT318可能通过作用于Syk525/6磷酸化而抑制血小板聚集。

Objective To observe the effect of PRT060318 （PRT318）, an inhibitor of spleen tyrosine ki- nase （Syk）, on human platelet aggregation and Syk phosphorylation in vitro. Methods The effect of PRT318 on platelet aggregation induced by collagen, adenosine diphosphate （ADP） and araehidonic acid （AA） was measured by turbidimetry. The phosphorylation of Syk525/6 was detected by Western blot. Results The Syk- specific inhibitor PRT318 suppressed collagen-induced platelet aggregation, while it did not have significantly inhibitory effect on ADP- or AA-induced platelet aggregation. The present data showed that PRT318 promoted the phosphory｜ation of Syk525/6 in collagen-induced platelets. PP2, tyrosine protein kinase （Src） inhibitor, inhibited collagen-induced platelet aggregation and decreased the level of Syk525/6 phosphorylation induced by collagen. PP2 also blocked PRT318-enhaneed Syk525/6 phosphorylation in collagen-treated platelets. Conclusions PRT318 significantly inhibits collagen-induced platelet aggregation and promotes collagen-induced phosphorylation of Syk525/6 locating in the downstream of glyeoprotein VI （GPVI） signal pathway, suggesting that PRT318 may inhibit platelet activation through regulating Syk525/6 phosphorylation.