PF4-coated E coli LPS mutants are recognized by anti-PF4/heparin IgG from sera of patients with HIT. (A) PF4-coated E coli LPS mutants ΔwaaC (KPM53) or ΔwaaA (KPM121) were incubated with sera of 3 patients with HIT known to contain anti-PF4/heparin antibodies, and bound IgG was affinity purified. Symbols represent reactivities of IgG antibodies eluted from PF4-coated E coli KPM53 (squares) or KPM121 (triangles); means are presented as horizontal lines. Binding of the purified antibodies to PF4/heparin complexes (first column) was inhibited by excess heparin (100 IU/mL unfractionated heparin [UFH], column 2), which disrupts PF4/heparin complexes. Antibodies did not react with PF4 alone (column 3). PF4-untreated bacteria served as control for unspecific binding of the antibodies to bacteria alone (column 4). (B) E coli K12 wild-type strain BW30270 and its isogenic ΔwaaA mutant KPM121 were labeled with FITC and preincubated with PF4 and additionally with heat-inactivated human serum containing anti-PF4/heparin IgG (preadsorbed with bacteria alone). After preincubation, the bacteria were subjected to phagocytosis. The figure shows the MFI multiplied by the percentage of FITC-positive polymorphonuclear leukocytes (PMNs) as a measure for bacterial phagocytosis. Data are mean ± SD of 4 different sera.