sulfate side groups is essential for a polyanion to react with PF4 and to create sites for antibody binding, arguing strongly against the possibility that HIT antibodies are specific to "compound epitopes" consisting partially of GAG and partially of peptide sequence at sites where the molecules making up the complex come into close contact. This observation, together with the finding that HIT antibodies fail to recognize heparin complexed with protamine (unpublished observation), excludes the possibility that they recognize a configuration of the sulfated sacchar-ide that is stabilized on binding to a small, positively charged, spherical protein.

It appears likely, therefore, that sites for antibody binding are created when linear polyanionic compounds bind to PF4 and alter its 3D configuration. Heparin-induced antibodies associated with HIT bind avidly to complexes formed between PF4 and heparin fragments attached by end-linkage to agarose beads but fail to recognize PF4 complexed with heparin molecules immobilized by multiple cross-linkages (Suh et al., 1998). Thus, another requirement for the formation of PF4-heparin complexes for which HIT antibodies are specific appears to be that the saccharide chain making up the heparin molecule must be in a flexible, relatively unconstrained state prior to reacting with PF4.

Although PF4-heparin complexes have not yet yielded to structural analysis, some informative data about the nature of PF4-heparin interaction and its effect on PF4 structure are available. Both bovine (St. Charles et al., 1989) and human (Zhang et al., 1994) PF4 tetramers have been crystallized and have similar structure. Each PF4 monomer consists of a COOH-terminal amphiphilic a-helix overlying a three-stranded antiparallel b-sheet, a structure typical of CXC chemo-kine family members (Luster, 1998). Two PF4 monomers associate side by side to produce a six-stranded antiparallel b-sheet, with overlying antiparallel a-helices (AB dimer). Each AB dimer associates with an identical CD dimer through surface interaction between the b-sheets. The elements of PF4 structure are shown schematically in Figure 2a.

Crystallographic studies have shown that both bovine (St. Charles et al., 1989) and human (Zhang et al., 1994) PF4 contain a ring of positively charged

FIGURE 2 (See color insert) (A) Computer-generated model (WebLab ViewerPro; Molecular Simulation Inc., San Diego, CA) of the human PF4 tetramer, based on the crystallographic coordinates. (B) AC dimer view of human PF4: the amino acid residues crucial for heparin binding are displayed. Abbreviation: PF4, platelet factor 4. Source: (A) from Zhang et al., 1994; (B), from Loscalzo et al., 1985; Mayo et al., 1995a.

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