Research Of Platelet Apoptosis Induced By Lipopolysaccharide/Peptidoglycan On Human In Vitro

Objective:Washed platelets were cultured with LPS or PGN in the presence or absence of blocking monoclonal antibody to human TLR4 or TLR2 in this research.The aim is to explore the effect of platelet apoptosis induced by LPS or PGN and,what’s more,the role of LPS-TLR4 or PGN-TLR2 pathway in this process; to provide evidence as basic research for the pathogenesis of septic thrombocytopenia;to offer potential therapeutic targets for improving the prognosis of patients with septic thrombocytopenia.Content:Part Ⅰ:Research of LPS induced platelet apoptosis and role of TLR4 in vitroFirst prepare washed platelets and divide them into two groups. The group of concentration gradient was cultured with multiple LPS concentrations,then the effect of platelet apoptosis induced by LPS was observed and the optimal concentration was chosen. For the group of TLR4 monoclonal antibody- blocking,thrombin was served as positive control to explore the change of platelet apoptosis induced by LPS in the presence or absence of TLR4 monoclonal antibody.Part Ⅱ:Research of PGN induced platelet apoptosis and role of TLR2 in vitro First prepare washed platelets and divide them into two groups. The group of concentration gradient was cultured with multiple PGN concentrations,then the effect of platelet apoptosis induced by PGN was observed and the optimal concentration was chosen. For the group of TLR2 monoclonal antibody- blocking,thrombin was served as positive control to explore the change of platelet apoptosis induced by PGN in the presence or absence of TLR2 monoclonal antibody.Methods:Firstly,prepare washed platelets and establish the reaction system in vitro. Then set up the concentration gradient groups(LPS/PGN) and the monoclonal antibody blocking groups(TLR4/TLR2 Mo Ab) respectively. Platelet apoptosis induced by thrombin was chosen as the positive control. Platelet PS evagination and Δ Ψdepolarization were observed by FCM.The expression of apoptosis related proteins was screened by Human Apoptosis Antibody Array. The expression of pro-apoptosis proteins Caspase-3,-8,Bax,Bak and anti-apoptosis protein Bcl-2 was detected by Western Blot.Results:Either LPS or PGN could induce platelet PS evagination and Δ Ψ depolarization in a concentration- dependent manner. Human Apoptosis Antibody Array showed us that LPS or PGN can change the expression of apoptosis-related proteins in platelets,which is characterized by the up-regulation of pro-apoptosis proteins and down-regulation of anti-apoptosis proteins mostly. But the result of Western Blot presented a different view which the expression of pro-apoptosis proteins(Caspase-3,-8,Bax,Bak) and anti-apoptosis proteins(Bcl-2) increased consistently.The reseach on the blocking of TLR4/TLR2 monoclonal antibody indicated that the pathways of TLR4-LPS/TLR2-PGN partially mediated platelet apoptosis induced by LPS/PGN at least.Conclusion:1. Either LPS or PGN could induce platelet apoptosis,which was manifested by PS evagination、Δ Ψ depolarization、increased Caspase-3 activity and up-regulation of apoptosis-related proteins Caspase-8、Bak、Bax and Bcl-2;2. The intrinsic mitochondrial pathway and exogenous death receptor pathway are all involved in the platelet apoptosis induced by LPS/PGN;3. TLR4 was involved in the platelet apoptosis induced by LPS, TLR4 monoclonal antibody could partially block the platelet apoptosis induced by LPS at least;TLR2 was involved in the platelet apoptosis induced by PGN, TLR2 monoclonal antibody could partially block the platelet apoptosis induced by PGN at least.