Binding of heparin‐dependent antibodies to PF4 modified by enoxaparin oligosaccharides: evaluation by surface plasmon resonance and serotonin release assay

Abstract

Summary. Background: The minimal structural requirements of low-molecular-weight heparins that determine the risk of developing heparin-induced thrombocytopenia (HIT) are not fully defined. Objectives: The ability of enoxaparin-derived oligosaccharides (OS) to induce platelet activation and exposure of platelet-factor 4 (PF4) epitopes recognized by antibodies developed in HIT was studied by surface plasmon resonance (SPR) and serotonin release assay. Results: Decasaccharides with ‡ 11 sulfate groups induced platelet activation in the presence of plasma from patients with confirmed HIT. Serotonin release of > 80% without full inhibition at 100 lg mL )1 was achieved with decasaccharides containing 14 or 15 sulfate groups, 2 dodecasaccharides and 2 tetradecasaccharides. An SPR method was developed using purified PF4 immobilized on carboxymethylated dextran. Antibodies from all HIT samples bound to PF4/heparin in SPR assays with resonance units (RU) ratio of 109-173 with HIT plasma vs. 88-93 with control plasma. RU ratios > 100 were measured when PF4 was pre-incubated with OS with ‡ 10 saccharide units and one octasaccharide containing 10 sulfate groups. RU ratios > 140, similar to those measured when PF4 was pre-incubated with unfractionated heparin or enoxaparin, were obtained with purified dodeca-and tetradecasaccharides. RU values strongly correlated with the number of sulfate groups in the decasaccharides tested (r = 0.93, P = 0.02). Conclusions: LMWHs with fragments > 10 saccharides and a large number of sulfate groups are more likely to be associated with a higher risk of HIT. These structure-activity relationships were independent of the ability of the OS to bind antithrombin.

Complex formation with nucleic acids and aptamers alters the antigenic properties of platelet factor 4

“…[25][26][27] The formation of epitopes on these complexes that are recognized by anti-PF4/heparin antibodies is dependent on critical structural features of the polyanions, for example, charge density, 26,28,29 length, 26,28 and charge clustering, such as those that occur on branched polysaccharides. 26 Nucleic acids express charged oxygens (O 2 ) of phosphate groups that are spaced 0.5 to 0.7 nm apart.…”

Mechanical prophylaxis is a heparin-independent risk for anti–platelet factor 4/heparin antibody formation after orthopedic surgery

“…16 Previous studies indicated that 11 to 12 saccharides constitute the minimal structure length necessary to complex with and induce antigenicity in PF4 to react with HIT antibodies. [21][22][23][24] In this context, fondaparinux, a pentasaccharide, is too small to strongly react with HIT antibodies. These observations suggest that fondaparinux would not be associated with antibody formation.…”

Heparin Mimetics: Their Therapeutic Potential

“…Thus, neither the monosaccharide composition nor the structure of the anion are determining features for PF4 binding. Studies with heparin showed that ≥10 saccharides and >13 sulfate groups are required for heparin fragments to bind PF4, trigger neoepitope exposure and HIT antibody binding, and it was concluded that charge density and oligosaccharide size were more important than a specific sulfation pattern for PF4 binding [ 152 ]. Given these findings it is probable heparin mimetics that broadly fit the criteria of ≥10 saccharides and >13 anions have the potential to bind PF4 in such a way so as to trigger drug induced thrombocytopenia.…”