Objective: To observe whether lipopolysaccharide ( LPS ) derived from Escherichia coli ( E.coli ) can induce apoptosis of murine platelets in vitro.
Methods: Washed platelet suspension was prepared and adjusted to the final concentration of 3×10(8)/mL. According to the difference in stimulants, samples were divided into control group ( non-calcium Tyrode buffer ), thrombin-treated group ( 1 U/mL final concentration and non-calcium TB ) and LPS in different concentrations treated groups ( 1, 10 and 100 μg/mL final concentration respectively and non-calcium TB ). To each specimental group corresponding stimulus was added and incubated 30 minutes at room temperature. Chemiluminescence was adopted to determine the concentration of adenosine triphosphate ( ATP ) and the activity of cysteinyl aspartate specific proteinase-3 ( caspase-3 ). The percentage of Annexin V positive platelets was determined by flow cytometry to reflect the level of phosphatidylserine ( PS ) exposure. Mean channel fluorescence ( MCF ) of platelets was determined by flow cytometry for reflecting the level of mitochondrial inner transmembrane potential (Δψm ) depolarization.
Results: Compared with control group, the ATP concentration in thrombin-treated group was decreased obviously [ relative light unit ( RLU ): ( 5.46±0.14 )×10(5) vs. ( 6.25±0.26 )×10(5), P < 0.05 ], Annexin V positive ratio [ ( 50.43±2.45 )% vs. ( 1.58±0.25 )%, P < 0.05 ] and caspase-3 activity [ RLU: ( 26.92±1.60 )×10(3) vs. ( 1.30±0.10 )×10(3), P < 0.05 ] were increased obviously, and platelets MCF was lowered significantly [ ( 8.32±0.58 )×10(4) vs. ( 13.05±1.10 )×10(4), P < 0.05 ], suggesting an increase in Δ ψm depolarization. After being treated with different concentrations of LPS, ATP concentration, Annexin V positive ratio and caspase-3 activity were increased obviously, platelet MCF was decreased obviously, suggesting Δψm depolarization was increased in a concentration-dependent manner. Compared with control group, 1 μg/mL LPS could increase Annexin V positive ratio [ ( 10.45±1.08 )% vs. ( 1.58±0.25 )%, P < 0.05 ], elevate caspase-3 activity [ RLU: ( 14.06±0.61 )×10(3) vs. ( 1.30±0.10 )×10(3), P < 0.05 ], and decrease MCF significantly [ ( 9.48±0.50 )×10(4) vs. ( 13.05±1.10 )×10(4), P < 0.05 ]. The ATP concentration, Annexin V positive ratio and caspase-3 activity reached maximum levels after the treatment with 100 μg/mL LPS, and they were higher obviously than those of the control group [ ATP ( RLU ): ( 7.00±0.03 )×10(5) vs. ( 6.25±0.26 )×10(5), Annexin V positive ratio: ( 55.35±2.42 )% vs. ( 1.58±0.25 )%, casepase-3 ( RLU ): ( 32.00±3.75 )×10(3) vs. ( 1.30±0.10 )×10(3), all P < 0.05 ], and platelets MCF reached trough levels, and they were obviously lower than those of the control group [ ( 4.69±0.55 )×10(4) vs. ( 13.05±1.10 )×10(4), P < 0.05 ].
Conclusions: E.coli LPS can induce an increase in ATP, PS exposure, Δψm depolarization and activity increase of caspase-3 on mouse platelet in vitro, which indicate that LPS can induce apoptosis of platelets in a concentration-dependent manner.