Nature Of The Epitopes Recognized By Hit Antibodies A The Role of Polyanion

The HIT antibodies fail to recognize PF4 or heparin alone but bind avidly to the PF4-heparin complex (Visentin et al., 1994). Antibody epitopes, therefore, could be composed of either combinatorial epitopes, consisting partly of heparin and partly of PF4, or conformational epitopes on the PF4 molecule induced by heparin binding. The epitopes could even be formed when two PF4 tetramers become closely apposed due to polysaccharide charge neutralization (Greinacher et al., 2006). Alternatively, a conformational change elsewhere on the PF4 molecule, created when the complex forms, could be targeted.

Heparin is a linear polyanion, and Maccarana and Lindahl (1993) have suggested that it binds to positively charged PF4 by nonspecific, electrostatic interactions rather than by specific oligosaccharide sequence recognition. However, Stringer and Gallagher (1997) have described a sequence on heparan sulfate consisting of a 9 kDa fragment, with sulfated domains at each end separated by a central, N-acetylated region, which may confer some specificity for PF4 binding. Regardless of whether PF4-heparin interaction is to some extent structure specific, non-GAG molecules can be substituted for heparin in detecting HIT antibodies. Kelton et al. (1994) found that highly sulfated polysaccharides, including heparan sulfate, pentosan polysulfate, and dextran sulfate, could be used, provided that they contained 1.0-1.5 sulfate groups per saccharide residue. Chondroitin sulfates A, B, and C, containing an average of only 0.5 sulfates per saccharide residue, were inactive. Highly sulfated but low molecular weight substances such as glucose-l,3,6-trisulfate, 1,2-cyclohexanediol disulfate, and heparin disaccharide, were likewise inactive. Greinacher and colleagues (1992, 1995) also characterized the structural requirements of polysaccharides active in generating HIT antibody epitopes. They showed that the b 1, 4-linkage between disaccharides, characteristic of heparin and other GAGs, was not essential, that heparin fractions containing fewer than 10 residues were unable to promote platelet serotonin release by HIT antibodies, and that branched glucan sulfates were more effective than linear glucan sulfates of the same molecular weight. Similarly, Amiral and coworkers (1995) found that the extent of polysaccharide sulfation is positively correlated with the ability to interact with PF4 in facilitating the binding of HIT antibodies.

It has been shown that in the presence of UFH, PF4 forms ultralarge complexes that are strongly antigenic, bind multiple IgG antibodies, and promote platelet activation (Rauova et al., 2005, 2006). However, recent data suggest that PF4 released from platelets can react directly with endogenous GAGs on the surface of platelets (Newman and Chong, 2000; Prechel et al., 2005; Rauova et al., 2006). Recognition of these complexes by high titer antibodies could explain "delayed HIT" occurring days or weeks after the last exposure to heparin in some individuals (Aster, 2005).

Studies conducted in our laboratory (Visentin et al., 2001) showed that UFH of bovine and porcine origin, as well as LMWH, formed complexes with PF4 that were recognized equally well by a panel of HIT antibodies. In studies with heparin fragments of known size, a length of at least 10 saccharide residues was required to form complexes with PF4 that reacted (weakly) with this antibody panel. For optimal antibody recognition, fragments containing at least 12 saccharide residues were required. Also, sulfated GAGs other than heparin (e.g., heparan sulfate) as well as non-GAG sulfated polysaccharides (e.g., fucoidan and dextran sulfate) behaved similarly to heparin in their ability to form antibody-binding complexes with PF4. However, the "heparinoid" anticoagulant danaparoid (Orgaran), a mixture of nonheparin low molecular weight GAGs having a low degree of sulfation, formed complexes that reacted with only about one-third of patient samples tested (Visentin et al., 2001). Our findings, together with those of Kelton, Greinacher, and Amiral already cited, indicate that the ability of GAGs and other sulfated polysaccharides to substitute for heparin in promoting platelet activation by HIT antibodies and to form complexes with PF4 to which the antibodies bind is directly related to the size and degree of sulfation of the polysaccharide.

To determine whether or not a polysaccharide structure is necessary for the formation of HIT antibody epitopes, we evaluated a series of linear, nonsacchar-ide, polyanionic compounds and found unexpectedly that polyvinyl sulfate, poly-vinyl sulfonate, polyvinyl phosphate, polyvinyl phosphonate, and polyanethole sulfonate all react with PF4 to produce complexes recognized by HIT-associated antibodies (Visentin et al., 2001) (Fig. 1). Thus, neither a saccharide chain nor


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