PF4 binds to Gram-positive (S aureus, S pneumoniae, and L monocytogenes) and Gram-negative bacteria (E coli and N meningitidis). (A) PF4 binding to Gram-positive and Gram-negative bacteria is dose-dependent and saturates at 20 μg/mL. Binding capacity for PF4 differs between bacterial strains, with E coli showing the highest binding capacity. Each bacterial strain was incubated with increasing concentrations of biotinylated hu-PF4 (solid symbols, all bacteria) or mu-PF4 (open symbols, E coli and S aureus). PF4 binding was detected with peridinin chlorophyll protein–Cy5.5-labeled streptavidin using flow cytometry and expressed as geometric mean fluorescence intensity (GMFI) multiplied by the percentage of labeled bacteria. Data are mean ± SD of 3 independent experiments. (B) PF4 binding to S aureus SA113spa is inhibited in a dose-dependent manner by heparin and dextran sulfate demonstrating charge dependency. S aureus SA113spa was incubated with buffer or with 20 μg/mL biotinylated hu-PF4 alone or in the presence of increasing concentrations of heparin or dextran sulfate (0, 12.5, 25, 50, and 100 μg/mL). PF4 binding was detected with peridinin chlorophyll protein–Cy5.5-labeled streptavidin using flow cytometry and expressed as GMFI multiplied by the percentage of labeled bacteria. Data are mean ± SD of 3 independent experiments.