Figure 2.

Heparin-dependent reactivity of monoclonal and polyclonal antibodies to PF4/heparin and PRT/heparin complexes. (A) Binding of KKO and HIT antibodies to PF4/heparin in the presence of excess heparin. KKO 2 ng/mL, HIT antibodies from patients (HIT 1-4, diluted 1:100), or normal plasma (1:100) were incubated in microtiter wells coated with PF4/heparin complexes either in buffer or in buffer supplemented with increasing concentrations of unfractionated heparin (0.1-100 U/mL). Concentrations in excess of 0.1 U/mL of heparin were associated with significant loss of KKO and HIT antibody binding. (B) Binding of ADA and PRT/heparin antibodies to PRT/heparin in the presence of excess heparin. ADA 100 ng/mL, patient-derived PRT/heparin antibodies (CPB1-4, diluted 1:500), or normal plasma (1:500) were incubated in microtiter wells coated with PRT/heparin complexes either in buffer or buffer supplemented with increasing concentrations of unfractionated heparin (0.1-100 U/mL). For CPB1, antibody binding was reduced with heparin concentrations ≥ 10 U/mL (78% decrease in binding). CPB2-4 showed significant binding despite excess heparin (>100 U/mL). Binding characteristics of antibodies are also shown in columnar format in supplemental Figure 1. All data shown are representative of 3 independent determinations.