One of the earliest animal models of HIT used the natural immune process of anti-idiotypic antibody production to invoke expression of HIT-IgG in mice (Blank et al., 1997, 1999). Mice immunized with HIT-IgG developed anti-idiotypic IgG that now recognized PF4-H. Unfortunately, this model has limited use, as the mice did not develop thrombosis, perhaps because murine platelets lack FcyRIIa.
Other investigators (Arepally et al., 2000) developed a murine monoclonal antibody, termed KKO, by immunizing mice with PF4-H. This murine IgG2bK monoclonal antibody mimics HIT-IgG, as it requires both PF4 and heparin to activate human platelets through their FcyRIIa. However, besides lacking FcyRIIa, mouse PF4 is not recognized by HIT-IgG or KKO. To overcome these problems, Reilly and colleagues (2001) produced transgenic mice that express both human FcyRIIa and human PF4. In these animals, addition of KKO caused thrombocyto-penia and death, including thrombosis of the lung vasculature. This murine model has proven useful to address immunological questions related to HIT. First, large macromolecular complexes are a necessary component in the development of HIT (Rauova et al., 2005). Second, a preexisting prothrombotic condition may influence the development of HIT. Mice fed a hypercholesterolemic diet had increased platelet and endothelial-cell activation and were predisposed to HIT to a greater extent than healthy, diet-fed syngeneic control mice (Reilly et al., 2006).
When platelet-activating (anti-CD9) IgG was administered to FcyRIIa trans-genic mice, more severe thrombocytopenia resulted, compared with a previously studied anti-mouse platelet (nonactivating) IgG (Taylor et al., 2000). Severe thrombosis, shock, and death developed in FcyRIIa transgenic mice crossed with FcRy-chain knockout mice. Moreover, splenectomy facilitated anti-CD9-mediated shock in FcyRIIa transgenic mice. The authors concluded that the clearance of antibody-sensitized platelets by phagocytic cells in the spleen may play a protective role in preventing thrombosis.
Unlike mice, primate platelets do possess FcyRIIa. Thus, a primate model for HIT may be feasible, as suggested by a recent report (Ahmad et al., 2000). The animals (Macaca mulatto) used do not express the human Arg-His polymorphism, perhaps explaining why less variability in platelet activation response to HIT-IgG was observed in these in vitro studies. The primate model may have value in evaluating therapeutic agents for HIT (Untch et al., 2002).
Monocytes and macrophages possess several different classes of FcyR (Table 1), and thus may play a part in influencing the frequency and severity of both thrombocytopenia and thrombosis in HIT. One role, discussed in the previous section, involves their potential to influence the balance between platelet activation and reticuloendothelial-mediated platelet clearance in HIT. Another function recently proposed for monocytes is that of contributing to the procoagulant state in HIT (a role posited previously for endothelial cells) (see Chapter 9). Pouplard and colleagues (2001) found that by adding HIT-IgG and PF4 (or PF4-H) directly to isolated monocytes or to whole blood, the monocytes produced TF, an effect that could be inhibited by high concentrations of heparin. Arepally and Mayer (2001) found that monocytes expressed surface TF when incubated with PF4 in the presence of either HIT-IgG or the HIT-mimicking murine monoclonal antibody, KKO. Because monocytes express sulfated proteoglycans on their surface, PF4 binding to monocytes can occur in the absence of added heparin. These studies raise the possibility that monocytes play an important role in the pathogenesis of the procoagulant state characteristic of HIT. Animal models suggest there may be a balance between platelet activation by HIT-IgG (predisposing one to thrombosis) and clearance of platelets by monocytes-macrophages (protecting somewhat against thrombosis). However, phagocytosis or NK cell destruction of antibody-sensitized platelets likely contribute to the thrombocytopenia since HIT is associated with an overrepresentation of FcyRIIIa-Val158 (Gruel et al., 2004), an FcyR with higher affinity for IgG1 and IgG3 (Table 1).
V. FcyRIIa POLYMORPHISMS IN DISEASE A. Determining the FcyRIIa Polymorphism
The FcyRIIa-Arg/His131 polymorphism was first identified on the basis of functional differences effected by anti-CD3 monoclonal antibodies of the murine IgGl subclass (Tax et al., 1983, 1984). Proliferation assays distinguished "high" and "low" responders relative to the effects of these anti-CD3 murine monoclonal antibodies on T-cell-dependent mitogenesis. Subsequently, individuals bearing the FcyRIIa-Arg131 phenotype were identified as the "high responders" and the functional differences between the two variants were later confirmed using other FcyRIIa-dependent assays such as erythrocyte antigen-rosetting, phagocytosis, and platelet activation (Clark et al., 1989; Warmerdam et al., 1991; Parren et al., 1992; Salmon et al., 1992). Murine monoclonal IgGl activate platelets of all three Arg/ His131 phenotypes, but the homozygous FcyRIIa-Arg131 variant requires less murine monoclonal antibody for platelet activation to occur.
The high-affinity binding of human IgG2 to FcyRIIa results when histidine is substituted at amino acid 131 of the mature protein (Warmerdam et al., 1991). FcyRIIa-His131 has a greater affinity for human IgG2 but a lower affinity for murine IgGl. Therefore, the terms high and low responder, used historically for the effects of murine monoclonal antibodies on Arg131 and His131 FcyRIIa phenotypes, respectively, is confusing, as the opposite reaction profile is observed with human IgG2. The high/low responder terminology has been largely replaced in favor of referring simply to the amino acid polymorphism.
The FcyRIIa-Arg/His131 variant polymorphism can be determined in three ways: (1) by functional assay such as T-cell-dependent proliferation or murine monoclonal antibody activation; (2) by specific binding using 41H16, a monoclonal antibody that binds exclusively to the FcyRIIa Arg131 variant; and (3) by molecular genotyping. Several DNA-based methods have been developed to genotype for the FcyRIIa-Arg/His131 nucleotide substitution (Clark et al., 1991; Osborne et al., 1994; Bachelot et al., 1995; Burgess et al., 1995; Jiang et al., 1996; Denomme et al., 1997; Flesch et al., 1998; Carlsson et al., 1998).
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