Binding isotherms of RLYB211 and RLYB212 to human and mouse platelets expressing HPA-1 alloantigens. (A-B) Washed human platelets from HPA-1a/a and HPA-1b/b individuals were incubated with serially diluted RLYB211, RLYB212, or normal human IgG (nhIgG) and incubated for 1 hour at room temperature. Bound antibodies were detected using a 1/200 dilution of FITC-conjugated goat anti-human IgG. Note the specificity of RLYB211 and RLYB212 for the HPA-1a alloantigen. Note also that RLYB211 is not able to saturate all available GPIIb-IIIa receptors, even at 10 mg/mL, because the HPA-1a–specific antibody is only a small fraction of the total polyclonal IgG present in RLYB211. (C) Platelets from the indicated species and having the indicated phenotypes were incubated with normal human IgG or RLYB211. Note that in addition to the specificity of RLYB211 for HPA-1a expressed on GPIIIa on the surface of human platelets, RLYB211 is also specific for the APLDQ form of mouse GPIIIa on the surface of humanized, but not WT, mouse platelets. (D) APLDQ platelets were incubated with increasing concentrations of polyclonal RLYB211 or the monospecific monoclonal antibody RLYB212, and antibody binding was quantified using flow cytometry to generate saturation-binding curves. Note that the addition of ∼4 IU/mL of either antibody is well below saturation and results in occupancy of <10% of the available GPIIb-IIIa receptors.