Heparin-induced thrombocytopenia/thrombosis (HIT/T) is a common complication of heparin therapy that is caused by antibodies to platelet factor 4 (PF4) complexed with heparin. The immune response is polyclonal and polyspecific, ie, more than one neoepitope on PF4 is recognized by HIT/T antibodies. One such epitope has been previously identified; it involves the domain between the third and fourth cysteine residues in PF4 (site 1). However, the binding sites for other HIT/T antibodies remain to be defined. To explore this issue, the binding site of KKO, an HIT/T-like murine monoclonal antibody, was defined. KKO shares a binding site with many HIT/T antibodies on PF4/heparin, but does not bind to site 1 or recognize mouse PF4/heparin. Therefore, the binding of KKO to a series of mouse/ human PF4 chimeras complexed with heparin was examined. KKO recognizes a site that requires both the N terminus of PF4 and Pro34, which immediately precedes the third cysteine. Both regions lie on the surface of the PF4 tetramer in sufficient proximity (within 0.74 nm) to form a contiguous antigenic determinant.The 10 of 14 HIT/T sera that require the N terminus of PF4 for antigen recognition also require Pro34 to bind. This epitope, termed site 2, lies adjacent to site 1 in the crystal structure of the PF4 tetramer. Yet sites 1 and 2 can be recognized by distinct populations of antibodies. These studies further help to define a portion of the PF4 tetramer to which self-reactive antibodies develop in patients exposed to heparin. IntroductionHeparin is the most common known cause of drug-induced immune thrombocytopenia, occurring in 1% to 3% of patients receiving unfractionated heparin. 1-3 A significant fraction of these patients develop limb-or life-threatening thromboses. 4,5 Heparininduced thrombocytopenia/thrombosis (HIT/T) is mediated by antibodies directed at complexes that form between heparin or other anionic mucopolysaccharides and platelet factor 4 (PF4) in plasma and on the surface of vascular cells. [6][7][8][9][10] Immune complexes composed of HIT/T antibodies and PF4/heparin bind to the surface of platelets and induce their activation by cross-linking Fc␥IIA receptors, [11][12][13] and bind to the surface of the endothelium and monocytes, 14-16 inducing procoagulant activity. 11,14 PF4 is a 70-amino acid, platelet-specific CXC chemokine in which the first 2 of the 4 conserved cysteine residues are separated by 1 amino acid residue. 17 PF4 has been sequenced 18 and cloned, 19 and its x-ray crystallographic structure has been defined ( Figure 1A). [20][21][22] PF4 exists in many biologic fluids primarily in the form of a tetramer with the 3 ␤-sheets of each subunit facing inwards, and the N and C termini lying on the surface of the molecule. The C termini are rich in lysines, which contribute to the circumferential ring of positive charges that form the interface between the PF4 tetramer and heparin. [23][24][25] The mechanism by which PF4/heparin complexes become antigenic is unknown. We have previously defined an ant...