TLR2-PI3K/Akt Signaling Pathway Involved in Platelet Activation Induced By Group B Streptococci

Abstract Background Platelets not only play an important role in the initiation of hemostasis and thrombosis, but also participate in the immune and inflammatory response. Most studies focus on the platelets-bacteria interaction and demonstrate that bacteria are capable of binding to, aggregating and activating platelets. Human platelets are reported to express several groups of TLRs, which participate in the inflammation process and monitoring host infection. Recent data from our laboratory demonstrated that Group B streptococci (GBS) could induce platelet aggregation and up-regulate the expression of CD62P and further study showed that platelet TLR2 might involve in the activation. GBS, or streptococcus agalactiae, is one of the most common cause of life-threatening sepsis, pneumonia and meningitis in neonates, pregnant women, the elderly and immunocompromised adults. Therefore, illuminating the mechanisms of GBS-induced platelet activation is important for providing the basis for platelets in defense against infection and immunity. Since increasing reports have shown that the PI3-K/Akt signaling pathway regulates platelet activation and hemostasis, so it is possible to research the TLR2 related signaling pathway. Methods 1. Platelets were from healthy volunteers (all genders, 25-52 years old) who had not taken any anti-platelet drugs (like aspirins, clopidogrel and abciximab) during the previous 30 days. GBS 639 were isolated from patient's venous blood. 2. Platelet aggregation, the expression of platelet CD62P and PAC-1 were used as the indicator of platelet activation. GBS-induced platelet aggregation was assayed by light transmission; platelet TLR2, CD62P and PAC-1 expression were determined by flow cytometry; AKT and AKT phosphorylation expression were determined by RT-PCR or western blot assay. In some experiments, platelets were pre-incubated with PI3-K specific inhibitors LY294002 or anti-TLR2 monoclonal antibody. 3. Statistical analysis: Data are reported as the mean ± SD. Treatment groups were compared with the appropriate control (s), and statistical significance was examined using the two-tailed t-test. Differences were considered significant when P <0.05. All values were analyzed using SPSS version 17.0 software. Results 1. Platelet activation and TLR2 protein expression: Platelet aggregation, surface expression of TLR2, CD62P and PAC-1 induced by GBS were increased significantly on the platelets upon activation with GBS 639. However incubated with anti-TLR2 monoclonal antibody, they all decreased. 2. PI3-K/Akt signaling pathway: Real-time PCR showed that the PI3-K and Akt mRNA expression levels were increased significantly in the platelets stimulated with GBS 639. Western blot results showed that of Akt phosphorylation triggered by GBS was occurred as early as 15 min and increased gradually to reach a peak at 2 h post-infection and no significant changes were observed in total Akt protein expression during the infection. However, the expression of p-Akt, platelet aggregation and surface expression of CD62P and PAC-1 induced by GBS were significantly inhibited in the presence of a PI3-K inhibitor LY294002. 3. The relationship between TLR2 and PI3-K/Akt signaling pathway in platelet activation: Platelet p-Akt expression levels induced by GBS were significantly decreased after the activity of platelet TLR2 was blocked by anti-TLR2 monoclonal antibody, and no significant changes in total Akt protein expression. Conclusions GBS induced platelet activation through the TLR2-PI3-K/Akt signaling pathway in human platelets. Disclosures No relevant conflicts of interest to declare.