L. Ziporen et al., DEFINING AN ANTIGENIC EPITOPE ON PLATELET-FACTOR-4 ASSOCIATED WITH HEPARIN-INDUCED THROMBOCYTOPENIA, Blood, 92(9), 1998, pp. 3250-3259
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.