By Hit Antibodies A Formation of Antigens Recognized by HIT Antibodies

The strong anionic character of heparin plays the predominant pathogenic role in formation of the HIT antigens. Heparin adheres to both cell surface-bound and free PF4, and may additionally increase free PF4 by its platelet-activating effects (Horne and Hutchison, 1998; Newman and Chong, 2000). Heparin binding to PF4 results in clustering of PF4 (Amiral et al., 1992; Rauova et al., 2005) forming linear, ridge-like, multimolecular complexes (Greinacher et al., 2006). From experiments using the pentasaccharide, it has been shown that antigenic PF4 complexes are formed not only by bridging of two PF4 molecules, but also when charge neutralization by polyanion allows positively charged PF4 tetramers to undergo close approximation (Greinacher et al., 2006). Some patients develop antibodies (HIT antibodies) against these complexes. Most HIT antibodies recognize noncontiguous conformational epitopes on the PF4 molecule (three distinct binding sites for HIT antibodies on the PF4-heparin complexes have been identified [Suh et al., 1998; Li et al., 2002]) that are produced when at least two PF4 tetramers are bound together by heparin (Horsewood et al., 1996; Newman and Chong, 1999; Greina-cher et al., 2006). Differences in the relative size, amount, and stability of the complexes may be responsible for the observed differences in immunogenicity (UFH > LMWH«fondaparinux) (Warkentin et al., 2005a) and clinically relevant cross-reactivity (UFH > LMWH»fondaparinux) (Savi et al., 2005; Warkentin et al., 2005a) (Fig. 3).

Differing optimal ratios of heparin and PF4 observed to form the HIT epi-topes are reported (Rauova et al., 2005; Greinacher et al., 2006), perhaps resulting from differences in heparin preparations used and in experimental design. The reported optimal molar ratios between heparin and PF4 are debatable because exact molar concentrations cannot be indicated for polydisperse mixtures of molecules such as heparin.

In a few cases, PF4 alone can be recognized by the HIT antibodies, as shown by the reaction of purified HIT antibodies with either PF4-heparin complexes or PF4 in the absence of heparin (Greinacher et al., 1994a; Newman and Chong, 1999; Amiral et al., 2000; Prechel et al., 2005). Since pretreatment of platelets with chondroitinase abolishes the platelet-activating effects of these heparin-independent antibodies (Rauova et al., 2006), endogenous platelet-associated GAGs are thought to take the role of heparin. This may also be an explanation for "delayed-onset HIT," where thrombocytopenia and thrombosis begin several days after heparin has been stopped (Warkentin and Kelton, 2001). One such patient developed high levels of antibodies against PF4-heparin complexes, together with thrombocytopenia and multiple thromboses, beginning about 1 wk after a single 5000-unit injection of heparin (Warkentin and Bernstein, 2003). Sera from patients with delayed-onset HIT activate platelets strongly in vitro even in the absence of added heparin. The most likely explanation is that these patients develop autoantibodies that recognize PF4 bound to platelet surface GAGs.

Potentially, other obscure factors can induce the HIT antigen. This hypothesis is strengthened by the observation that on exceptionally rare occasions, patients can be identified with a thrombocytopenic, prothrombotic disorder that serologically mimics HIT but in whom no previous heparin exposure can be identified (Warkentin et al., 2006a). A related observation could be the finding

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