PF4 interaction with isolated lipid A relies on the phosphate groups. (A) PF4 binds dose-dependently to lipid A but not to LPS, Kdo₂-lipid A, or to Kdo alone. Binding of PF4 to MPLA (mono-(4′-P)-phosphoryl lipid) is reduced compared with binding to bisphosphorylated lipid A (Lipid A). Binding of biotinylated PF4 (0.06, 0.13, 0.25, 0.5, 1, 2, and 4 μg/mL) to immobilized LPS, Lipid A, Kdo₂-lipid A, MLPA, or Kdo (50 μg/mL) was detected with peroxidase-conjugated streptavidin followed by addition of tetramethylbenzidine. Data are mean OD ± SD of 3 independent experiments. (B) Binding of PF4 to Lipid A is inhibited by BPI or polymyxin B sulfate. Binding of biotinylated PF4 (2 μg/mL) in the presence of BPI or polymyxin (0.5, 1, 2, 4, and 8 μg/mL each) or buffer to immobilized Lipid A (50 μg/mL) was detected with peroxidase-conjugated streptavidin followed by addition of tetramethylbenzidine. Data are mean OD ± SD of 3 independent experiments. *P < .05 versus PF4-biotin + buffer. **P < .01 versus PF4-biotin + buffer. (C) Preincubation of PF4 with LPS, lipid A, Kdo₂-lipid A, but not Kdo alone, inhibits PF4 binding to the E coli KPM121 displaying only lipid IV_A on its surface, whereas the inhibitory effect of MPLA is reduced. PF4-biotin (40 μg/mL) was preincubated with Kdo, LPS, Kdo₂-lipid A, Lipid A, MPLA (each with 200 μg/mL), or buffer before assessing PF4 binding to E coli KPM121 by flow cytometry. The results are expressed as geometric mean fluorescence intensity (GMFI) multiplied by the percentage of labeled bacteria. Data represent mean ± SD of 3 independent experiments. **P < .01 versus PF4-biotin preincubated with buffer. ***P < .001 versus PF4-biotin preincubated with buffer.