Thrombin induces TLR4 expression on human platelets via the PAR/PLC pathway.

<p>(A) Washed human platelets were treated with 0.4 U/mL thrombin at 37°C for 20 min, and the level of TLR4 on the surface of platelets was determined by flow cytometry. (B) Washed human platelets were treated with 0.1–0.4 U/mL thrombin at 37°C for 20 min and analyzed by flow cytometry for the surface level of TLR4 (n = 3). (C) The total and membrane protein fraction were extracted, and the TLR4 levels were further confirmed by western blot analysis and detected with anti-TLR4 antibody. α-tubulin served as the loading control in this assay. The bar graph showed the quantification of western blot analysis using densitometry. (D) Human platelets were directly treated with SFLLRN, AYPGKF, or SFLLRN plus AYPGKF at 37°C for 20 min. The level of surface TLR4 was determined by flow cytometry. (E) Washed human platelets were pretreated with U73122 or Y27632 at 28°C for 60 min followed by 0.4 U/mL thrombin treatments at 37°C for 20 min, and the surface level of TLR4 on the platelets was determined by flow cytometry. The m-3M3FBS was pretreated at 28°C for 60 min, than incubate at at 37°C for 20 min. The data represented the results of 5 independent experiments (mean ± SD; *<i>p</i><0.05).