DEFINING AN ANTIGENIC EPITOPE ON PLATELET-FACTOR-4 ASSOCIATED WITH HEPARIN-INDUCED THROMBOCYTOPENIA

Heparin induced thrombocytopenia (HIT) is a potentially serious compli cation of heparin therapy. Antibodies to platelet factor 4 (PF4)/hepar in complexes have been implicated in the pathogenesis of this disorder , but the antigenic epitope(s) on the protein have not been defined. T o address this issue, we studied the binding of HIT antibodies to a se ries of recombinant proteins containing either point mutations in PF4 or chimeras containing various domains of PF4 and the related protein, neutrophil activating peptide-2 (NAP-2). Serum samples from 50 patien ts with a positive C-14-serotonin release assay (C-14-SRA) and a clini cal diagnosis of HIT and 20 normal controls were studied. HIT antibodi es reacted strongly with wild-type (WT) PF4/heparin complexes, but rea cted little, if at all, with NAP-2/heparin complexes (optical density [OD](405) = 2.5 and 0.2, respectively). Alanine substitutions at three of the four lysine residues implicated in heparin binding, K62, K65, and K66, had little effect on recognition by HIT antibodies (OD405 = 2 .2, 2.8, and 2.0, respectively), whereas an alanine substitution at po sition K61 led to reduced, but still significant binding (OD405 = 1.0) . Similar studies involving chimeras between PF4 and NAP-2 localized a major antigenic site to the region between the third and fourth cyste ine residues for more than half of the sera tested. This site appears to involve a series of amino acids immediately after the third cystein e residue beginning with P37. Thus our studies suggest that whereas th e C-terminal lysine residues of PF4 are important for heparin binding, they do not comprise a critical antigenic site for most HIT antibodie s. Rather, we propose that maintaining a region near the third cystein e residue of PF4, distal from the proposed heparin-binding domain, is required to form the epitope recognized by many HIT antibodies. (C) 19 98 by The American Society of Hematology.