Functionality of PF4-specific rAbs. (A) PF4 antibody (Ab) complex detection assay. Schematic representation showing only minor binding of IgG1 anti-PF4 rAbs to FcγRIIa due to low affinity (top left) and high binding responses of immune complexes formed on the addition of PF4, thereby increasing avidity (top right). BLI assay with immobilized FcγRIIa, which is dipped into wells containing anti-PF4 rAbs without PF4 (bottom left panel) or with PF4 (right panel), followed by a dissociation step in empty buffer. (B) PF4-induced platelet activation assay (PIPAA). PF4 + buffer compared with buffer alone (left), or PF4 + buffer compared with PF4 + buffer + FcγRIIa-blocking monoclonal antibody (middle). Heparin-induced platelet activation assay (right). Heparin (2 U/mL) compared with excess heparin (1000 U/mL) or heparin + FcγRIIa-blocking monoclonal antibody. Each rAb was tested in quadruplicate with 4 different donor platelet suspensions. Each bar represents the mean with SD of the quadruplicate tests. The comparison was performed with platelets from the same donors. The time to platelet activation (aggregation, visual readout every 5 minutes) is displayed. A time of ≤45 minutes is considered positive, and >45 minutes is considered negative. Positive control (pos contr): pooled human sera with known strong positive (5- or 10-minute) results in PIPAA. Negative control (neg contr): inert serum from healthy control with blood group AB.