Disruption of PF4/H multimolecular complex formation with a minimally anticoagulant heparin (ODSH)

Abstract

Recent studies have shown that ultra-large complexes (ULCs) of platelet factor 4 (PF4) and heparin (H) play an essential role in the pathogenesis of heparin-induced thrombocytopenia (HIT), an immune-mediated disorder caused by PF4/H antibodies. Because antigenic PF4/H ULCs assemble through non-specific electrostatic interactions, we reasoned that disruption of charge-based interactions can modulate the immune response to antigen. We tested a minimally anticoagulant compound (2-O, 3-O desulfated heparin, ODSH) with preserved charge to disrupt PF4/H complex formation and immunogenicity. We show that ODSH disrupts complexes when added to pre-formed PF4/H ULCs and prevents ULC formation when incubated simultaneously with PF4 and UFH. In other studies, we show that excess ODSH reduces HIT antibody (Ab) binding in immunoassays and that PF4/ODSH complexes do not cross-react with HIT Abs. When ODSH and unfractionated heparin (UFH) are mixed at equimolar concentrations, we show that there is a negligible effect on amount of protamine required for heparin neutralisation and reduced immunogenicity of PF4/UFH in the presence of ODSH. Taken together, these studies suggest that ODSH can be used concurrently with UFH to disrupt PF4/H charge interactions and provides a novel strategy to reduce antibody mediated complications in HIT.

Figures

Figure 1. Comparison of ODSH and UFH with respect to thrombin generation and PF4 binding

(A) Thrombin generation assay. UFH or ODSH were incubated with antithrombin, thrombin and chromogenic substrate in buffer containing 50mM Tris, 150mM NaCl and 0.5% bovine serum albumin, pH 7.4. The amount of UFH or ODSH required to inhibit 50% of thrombin was calculated. Each data point represents mean ± SD of duplicate wells and is representative of three independent experiments. (B) Competition of labeled H (35S) with unlabeled UFH or ODSH in reaction buffer containing 50 mM Tris and 130 mM NaCl, pH 7.3. Binding was assayed as bound 35S. Each data point corresponds to mean ± SD of duplicate wells and is representative of two independent experiments.

Figure 2. Changes in light absorbance (A) and surface charge (B) of PF4 with increasing amounts of UFH or ODSH

PF4 (100µg/ml) was incubated with increasing amounts of UFH or ODSH in H2O and changes in absorbance (A) and zeta potential (B) were measured. Each data point corresponds to an individual measurement and is representative of three independent experiments.

Figure 3. Disruption of PF4/H or PF4/ODSH complexes by excess UFH or ODSH

PF4/UFH or PF4/ODSH complexes were formed in H20 as described in methods and size measured by PCS. In Runs A–D, complexes were first formed for 15 minutes and subsequently incubated with increasing amounts of UFH or ODSH as described for another 15 minutes and size measured by PCS. In Run E, PF4 was mixed concurrently with UFH and ODSH and complex size measured after 30 minutes. Each data point corresponds to an individual measurement and is representative of two independent experiments.

Figure 4. Binding of HIT antibodies

(A) Effects of excess ODSH on HIT antibody binding to PF4 antigenic complexes. HIT antibodies (1–5) or control subjects (1–2) were tested in a commercial HIT ELISA in the presence of increasing amounts of ODSH (0.5–10 µg/mL) in diluent buffer supplied by the manufacturer. Each data point corresponds to mean ± SD of duplicate wells and is representative of three independent experiments (B) Cross-reactivity of HIT antibodies with PF4/ODSH complexes. HIT antibodies (6–10), control subject and KKO, a murine HIT-like monoclonal antibody, were incubated with wells coated with human PF4 and increasing amounts of ODSH (0.4–3.2 µg/mL) and binding was compared to wells coated with huPF4/UFH. Each data point corresponds to mean ± SD of duplicate wells and is representative of two independent experiments.

Figure 5. Protamine neutralization of UFH or UFH+ODSH

UFH (0.5 U/mL) was incubated with or without increasing concentrations of ODSH (2.6, 5.2 and 10.4 µg/mL) and a protamine (PRT) neutralization assay was performed as described in methods. The amount of PRT required to inhibit 50% of thrombin generation was calculated as IC50. Each data point corresponds to mean ± SD of duplicate wells and is representative of two independent experiments.

Figure 6. Immunization with mPF4/H in the presence or absence of ODSH

mPF4 was mixed with H (5 U/mL) or H with equimolar amounts of ODSH (26.2 µg/mL) and complexes were injected into animals daily for five days. Significant differences were seen in mice injected with mPF4/H v. mPF4 (H+ODSH) (p