The immune response in heparin-induced thrombocytopenia is initiated by and directed to large multimolecular complexes of platelet factor 4 (PF4) and heparin (H). We have previously shown that PF4:H multimolecular complexes assemble through electrostatic interactions and, once formed, are highly immunogenic in vivo. Based on these observations, we hypothesized that other positively charged proteins would exhibit similar biologic interactions with H. To test this hypothesis, we selected 2 unrelated positively charged proteins, protamine (PRT) and lysozyme, and studied H-dependent interactions using in vitro and in vivo techniques. Our IntroductionHeparin-induced thrombocytopenia (HIT) is an immune-mediated disorder caused by antibodies that recognize multimolecular complexes of platelet factor 4 (PF4), a positively charged platelet protein, and heparin (H), a negatively charged carbohydrate. We, and others, have shown that PF4 and H complexes assemble primarily through nonspecific electrostatic interactions governed by principles of colloidal chemistry. [1][2][3][4][5] In colloidal systems, molecules of opposite charge "aggregate" or grow in size due to effects of charge neutralization. Particle interactions are frequently dependent on stoichiometric ratios of the 2 compounds, with the largest complexes occurring at molar ratios of the compounds leading to charge neutralization. When either compound is in molar excess, charge restabilization occurs and repulsive forces predominate, leading to reduced complex size and/or complex disassembly.Studies to date indicate that PF4/H multimolecular complex formation is central to the pathogenesis of HIT. The characteristic bell-shaped curve seen with HIT antibody binding over a range of H concentrations coincides with H-dependent formation of multimolecular complexes. 2,3 HIT antibody binding, as gauged by serologic assays or functional studies of platelet activation, is optimal when multimolecular complexes form at or near equimolar ratios of PF4:H. However, antibody binding is markedly reduced with increasing H concentrations, a phenomenon that can be directly attributed to loss of complex formation. [2][3][4] Recent studies from our laboratory indicate that similar H-dependent changes affect the immunogenicity of PF4/H complexes in vivo. 5,6 Our studies demonstrate that PF4/H complexes are immunogenic over a certain range of H concentrations associated with multimolecular complex formation and that the immune response is attenuated when PF4 or H is given alone or when H is in molar excess of PF4. 5 H and H-like molecules bind several positively charged proteins in addition to PF4. 7 These H-binding proteins (HBPs) are structurally and functionally diverse, and include, to name a few, nuclear proteins (protamine), enzymes (C1 esterase and lysozyme), adhesion molecules (fibronectin and vitronectin) growth factors (fibroblast growth factor), and lipid-binding proteins (apolipoprotein E and lipoprotein lipase). To date, it appears that a majority of HBP-H interactions a...

Platelet factor 4 (PF4)/heparin antibody, typically associated with heparin therapy, is reported in some heparin-naive people. Seroprevalence in the general population, however, remains unclear. We prospectively evaluated PF4/heparin antibody in approximately 4,000 blood bank donors using a commercial enzyme-linked immunosorbent assay for initial and then repeated (confirmatory) testing. Antibody was detected initially in 249 (6.6%; 95% confidence interval [CI], 5.8%-7.4%) of 3,795 donors and repeatedly in 163 (4.3%; 95% CI, 3.7%-5.0%) of 3,789 evaluable donors. “Unconfirmed” positives were mostly (93%) low positives (optical density [OD] = 0.40-0.59). Of 163 repeatedly positive samples, 116 (71.2%) were low positives, and 124 (76.1%) exhibited heparin-dependent binding. Predominant isotypes of intermediate to high seropositive samples (OD >0.6) were IgG (20/39 [51%]), IgM (9/39 [23%]), and indeterminate (10/39 [26%]). The marked background seroprevalence of PF4/heparin antibody (4.3%-6.6%) with the preponderance of low (and frequently nonreproducible) positives in blood donors suggests the need for further assay calibration, categorization of antibody level, and studies evaluating clinical relevance of “naturally occurring” PF4/heparin antibodies.

1435 Platelet Factor 4 (PF4)/heparin (H) multimolecular complexes initiate an immune response that can ultimately lead to complications of Heparin-Induced Thrombocytopenia (HIT), a life-threatening prothrombotic disorder. We have previously shown that PF4:H multimolecular complexes assemble through non-specific electrostatic interactions and that other unrelated positively-charged proteins such as protamine (PRT) and lysozyme (Lys) exhibit similar biophysical interactions with heparin (ASH 2009; abstract # 1316). In these earlier studies, we showed that PRT/H and Lys/H, like PF4/H, show heparin-dependent binding over a range of heparin concentrations and that formation of multimolecular complexes occurs at distinct stoichiometric ratios (PRT/H at 3:1 and Lys/H at 5:1 molar ratios). We now extend these observations in vivo to show relevance to human disease. Using a murine immunization model, we show that mice injected with PRT/H and Lys/H multimolecular complexes, but not PRT alone, Lys alone or buffer, develop antigen-specific immune responses. In additional studies, we show that the immune response to PRT/H or Lys/H shares important biologic similarities with the humoral response to murine (m) PF4/H multimolecular complexes. Specifically, we demonstrate that antibody formation to PRT/H and Lys/H is heparin-dependent (occurs optimally at certain stoichiometric ratios) dose-dependent (requires threshold amounts of multimolecular complexes) and shows serologic transience. To demonstrate the clinical relevance of our findings, we examined patients undergoing cardiopulmonary bypass (CPB) for development of PRT/H antibodies. For these studies, we assayed the plasma from healthy subjects (n=45) and patients undergoing CPB (n=15) at three time points {baseline (BL), 5 days (5D) and 30 days (30D) after CPB} for the presence of PRT/H antibodies. As shown Figure 1A, plasma from normal subjects and patients undergoing CPB patients at BL and D5 displayed minimal reactivity in the PRT/H ELISA. However, by 30D, we observed that 4/15 patients (27%) developed significantly elevated levels of antibodies to PRT/H as compared to normals, or their respective samples obtained at baseline or 5D after surgery. Seropositive patients (filled symbols, n=4) as compared to seronegative patients (open symbols, n=3) recognized PRT/H and to some extent, PRT alone, but did not cross-react with other antigens including PRT/H, BSA, Lys, Lys/H or human PF4/H, Figure 1B; p<0.001). To identify the mechanism by which protein/heparin multimolecular complexes triggered immune activation, we incubated murine dendritic cells from non-immunized C57Bl/6 mice with heparin or buffer, protein (mPF4, PRT or Lys), or protein/H complexes and measured IL-12, a marker of dendritic cell activation. As shown in Figure 1C, we demonstrated that IL-12 levels were significantly increased in wells containing protein/H complexes as compared to wells containing uncomplexed protein, buffer or heparin. Taken together, these studies indicate that heparin significantly alters the biophysical and biological properties of positively-charged compounds through formation of macromolecular complexes that lead to dendritic cell activation and trigger immune responses in vivo. Disclosures: Arepally: Glaxo Smith Kline: Speakers Bureau; Paringenix: Research Funding; University Of New Mexico: Patents & Royalties; Amgen: Speakers Bureau.