Mutagenesis studies imply a role for RKH and Y5motifs in platelet activation by PA clones. (A) HCDR3 sequences of clones studied. Critical amino acids in RKH and Y5 motifs are bolded. (B) Ala substitution for basic amino acids at positions 106, 109, and 112 of the RKH motif in PA clone HIT1P4B1 impaired its ability to bind PF4/H and to activate PF4-treated platelets. (C) Ala substitutions at positions 116 to 118 and 116 to 121 of the Y5 motif of PA clone HIT1P3D4 impaired its ability to bind to PF4/H and to activate PF4-treated platelets. (D) An R106K substitution in PA clone HIT2P4D5 abolished its ability to activate PF4-treated platelets but had only minimal effect on PF4/H binding. (E) A K106R substitution in NA clone HIT2P4D2 conferred platelet-activating function on the clone, while preserving PF4/H binding. (F) A D110A substitution in NA clone HIT5P7B10 significantly enhanced its platelet-activating ability and markedly increased its avidity for PF4/H. Concentrations of clonal IgG used in each reaction mixture are shown beneath B through F. Data shown are representative of 3 independent experiments for each clone and its mutant. Error bars represent mean ± SD of 2 replicates within each experiment. P values were calculated by 2-way analysis of variance. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001.
