Cellular Fn-EDA+ potentiates platelet aggregation through TLR4. (A) Representative immunoblots showing interaction between cellular Fn-EDA+ and TLR4. Proteins in platelet lysates from Fn-EDA^+/+ or Fn-EDA^−/− mice were immunoprecipitated with anti-Fn or anti-TLR4 antibodies or control IgG and immunoblotted using anti-TLR4 antibodies. (B) Representative tracings and corresponding bar diagrams showing aggregation responses of WT or TLR4^−/− platelets to thrombin (0.02 U/mL) in the presence of 40 µg/mL of either human pFn or cFn. Tracing 1 represents WT washed platelets treated with pFn; tracing 2 represents TLR4^−/− washed platelets treated with pFn; tracing 3 represents WT washed platelets treated with cFn; and tracing 4 represents TLR4^−/− washed platelets treated with cFn. Data are presented as mean ± SEM. N = 4 to 5 mice per group. (C) Representative immunoblots and corresponding bar diagrams showing expression of phospho-NF-κB p65 and phospho-IKK α/β relative to β-actin in lysates of thrombin (0.02 U/mL) aggregated (4 minutes) WT and TLR4^−/− platelets in the presence of either pFn or cFn. Data are presented as mean ± SEM. N = 3 mice per group. (D) Bar diagrams showing aggregation responses in PRP induced by adenosine 5′-diphosphate (ADP; 4 µM) or collagen (5 µg/mL). Data are presented as mean ± SEM. N = 5 to 6 mice per group.