血小板TLR4表达介导LPS诱导的血小板活化

本研究探讨人血小板Toll样受体4（toll—like receptor4，TLR4）表达及其在细菌内毒素脂多糖（lipopolysaccsharide，LPS）诱导血小板活化中的作用。对15例健康人肘静脉抽血各20ml，制备富含血小板血浆（PRP）和血小板悬液，在有或无凝血酶作用的基础上加入LPS（0．2μg/ml）孵育30分钟，分别用流式细胞术和免疫印迹（Western blot）方法检测LPS作用前后血小板TLR4、CD62P和CD40L的表达。结果表明：流式细胞术检测静止血小板TLR4表达为25．44％，凝血酶刺激后明显升高（32．34％，P〈0．05），LPS继续作用后TLR4的表达进一步增高（39．16％，P〈0．01）；在无凝血酶刺激下，LPS作用血小板后TLR4的表达接近于凝血酶刺激后结果。Westernblot方法检测结果与流式细胞术相符；血小板CD62P和CD40L表达（流式细胞术）在静止血小板分别为6．39％和2．45％，凝血酶刺激后明显增高，分别为42．68％和14．8％，LPS进一步刺激后表达率进一步升高，分别为63．03％和13．94％，均明显高于仅用凝血酶的刺激组（P〈0．001）；抗TLR4抗体显著抑制了LPS诱导的血小板TLR4、CD62P、CD40L表达，但不影响凝血酶诱导的血小板膜CD62P、CD40L表达。结论：人血小板能够表达功能性TLR4，它直接参与了机体针对细菌的固有免疫反应。

Toll-like Receptor 4 Expression Mediates the Activation of Platelets Induced by LPS

The study was aimed to investigate the expression of Toll-like receptor 4 （TLR4） on platelets and to determine whether platelet TLR4 involves in its activation induced by lipopolysaccsharide （LPS）. Human platelet-rich plasma （PRP） and platelet suspension obtained from 15 healthy individuals pretreated with a concentration of 0.2μg/ml of LPS in the presence or absence of thrombin （1 U/ml） for 1 hour. The expressions of TLR4 ,CD62P （P-select） and CD40L on platelets were detected by flow cytometry, and platelet TLR4 expression was further determined by Western blot analysis. The results indicated that the percentage of TLR4-positive platelets induced by thrombin was increased by 32.34% compared with the resting platelets （ 25.44%, p 〈 0.05 ）. TLR4 expression on platelets treated with LPS was remarkably elevated in the presence or absence of thrombin. However, the expression level of the former was much higher than that of the latter and thrombin stimulation alone （p 〈 0.05 ）. Moreover, the similar results were found in Western blot analysis. Synchronously, expressions of CD62P and CD40L on resting platelets were 6.39% and 2.45%, they were also markedly increased when treated with thrombin （42.68% and 14.8% ） and LPS respectively, and the increases of expression of CD62P and CD40L were more significant when stimulated with both LPS and thrombin （63. 03% and 13.94% ）. Although anti-TLR4 antibody inhibited significantly the increase of TLR4, CD62P and CD40L on platelets induced by LPS, which did not affect their increase induced by thrombin. In conclusion, the evidence has been shown that functional TLR4 can be expressed on human platelets. It may involve in platelet activation as an important mediator of LPS-induced CD62P and CD40L expressions on platelets.