Anti-platelet factor 4/heparin antibodies in orthopedic surgery patients receiving antithrombotic prophylaxis with fondaparinux or enoxaparin

Abstract

Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating IgG antibodies that recognize platelet factor 4 (PF4) bound to heparin. Immunogenicity of heparins differs in that unfractionated heparin (UFH) induces more anti-PF4/heparin antibodies than low-molecular-weight heparin (LMWH) and UFH also causes more HIT. Fondaparinux, a synthetic anticoagulant modeled after the antithrombin-binding pentasaccharide, is believed to be nonimmunogenic. We tested 2726 patients for anti-PF4/heparin antibodies after they were randomized to receive antithrombotic prophylaxis with fondaparinux or LMWH (enoxaparin) following hip or knee surgery. We also evaluated in vitro cross-reactivity of the IgG antibodies generated against PF4 in the presence of UFH, LMWH, danaparoid, or fondaparinux. We found that anti-PF4/heparin antibodies were generated at similar frequencies in patients treated with fondaparinux or enoxaparin. Although antibodies reacted equally well in vitro against PF4/UFH and PF4/LMWH, and sometimes weakly against PF4/danaparoid, none reacted against PF4/fondaparinux, including even those sera obtained from patients who formed antibodies during fondaparinux treatment. At high concentrations, however, fondaparinux inhibited binding of HIT antibodies to PF4/polysaccharide, indicating that PF4/fondaparinux interactions occur. No patient developed HIT. We conclude that despite similar immunogenicity of fondaparinux and LMWH, PF4/fondaparinux, but not PF4/LMWH, is recognized poorly by the antibodies generated, suggesting that the risk of HIT with fondaparinux likely is very low.

Figure 1.

Anti–PF4/heparin antibody formation in patients receiving fondaparinux or enoxaparin after orthopedic surgery (current status analysis). Data are combined for patients undergoing knee and hip replacement. (A) Anti–PF4/heparin antibodies of IgG class. There is no significant difference between the groups (P = .86). (B) All anti–PF4/heparin antibodies. There is no significant difference between the study drug groups (P = .24).

Figure 2.

Ratio of antibody binding to PF4/polysaccharide complexes compared to PF4 alone by fluid-phase EIA. Results of fluid-phase EIA testing for sera from 15 patients who formed anti–PF4/heparin-IgG antibodies (detected using solid-phase EIA) while receiving enoxaparin (n = 6, •) or fondaparinux (n = 9, ○). The data are expressed as ratios of binding to PF4 in the presence of polysaccharide (UFH, 0.6 IU/mL; LMWH, 0.5 anti-Xa U/mL; danaparoid, 0.1 anti-Xa U/mL; and fondaparinux, 0.1, 0.4, 1.2, and 10.0 μg/mL) over the baseline (buffer). Horizontal bars indicate medians. * indicate the 4 samples that tested positive (in the presence of UFH) in the platelet activation assay. For comparison, results are also shown for 15 patients with clinical HIT (▪). Statistically significant increases in reactivity (null hypothesis, mean ratio of OD [presence of drug]/OD [presence of buffer] = 1) for the 15 sera obtained from patients in the orthopedic trials were observed for UFH (P = .003), LMWH (P < .001), danaparoid (P = .002), but not with fondaparinux at any concentration (P > .05). Whereas 14 of 15 sera from patients in the orthopedic trials exhibited more than 2-fold greater reactivity than baseline against PF4/LMWH, none reacted similarly against PF4/fondaparinux (P < .001 by the McNemar test, 2-tailed).

Figure 3.

Inhibition of anti–PF4/polysaccharide reactivity by fondaparinux. (Top) Solid-phase anti–PF4/UFH EIA. Mean (± SEM) reactivity of 5 HIT sera is shown in the absence (buffer) and presence of increasing concentrations of fondaparinux (100, 200, 400, or 1000 μg/mL, final). Progressive inhibition of reactivity is seen that is significant at all concentrations of fondaparinux. As expected, high heparin (100 IU/mL) also inhibited reactivity. (Bottom) Fluid-phase anti–PF4/UFH (•) and anti–PF4/LMWH (○) EIAs. Mean (± SEM) reactivity of 5 HIT sera is shown in the absence (buffer) and presence of increasing concentrations of fondaparinux (100, 200, 400, or 1000 μg/mL, final). Progressive inhibition of reactivity is seen that is significant at all concentrations of fondaparinux. High heparin (100 IU/mL) and enoxaparin (100 IU/mL) concentrations also inhibited reactivity.