Affinity purification of heparin-dependent antibodies to platelet factor 4developed in heparin-induced thrombocytopenia: biological characteristics and effects on platelet activation

Antibodies to heparin platelet factor 4 (H-PF4) complexes were purified fro m the plasma of three patients with heparin-induced thrombocytopenia (HIT) using affinity chromatography. From each plasma, the largest amount of anti bodies was eluted with 2 M NaCl at pH 7.5 (peak 1) and the remainder was ob tained using 0.1 M glycine/0.5 M NaCl at pH 2.5 (peak 2). In an enzyme-link ed immunosorbent assay (ELISA), we then showed that each patient had develo ped antibodies to PF4 displaying different characteristics. In patient 1, p eak 1 IgG reacted almost exclusively with H-PF4 complexes, whereas peak 2 I gG had similar reactivity with PF4 whether or not heparin was present, Pati ent 2 expressed a mixture of IgA, IgM and IgG and both fractions bound to P F4 alone or to H-PF4 complexes. Finally. IgG in patient 3 only bound to H-P F4 and was unreactive with PF4 alone. Using [C-14]-serotonin release assays , the antibodies developed in the three patients and exhibiting the stronge st ability to activate platelets with heparin were those having the highest affinity to H-PF4. These results strongly support the hypothesis that HIT antibodies to PF4 are heterogeneous regarding their affinity and specificit y for target antigens and this may greatly influence their ability to activ ate platelets and their pathogenicity.