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

Abstract

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 100μgmL-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. © 2012 International Society on Thrombosis and Haemostasis.