title: 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.
  
  
  OBJECTIVE
  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 μg 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.
authors.affiliation: Department of Hematology-Hemostasis, University Hospital of Tours GICC UMR 6239 CNRS, University Francois Rabelais, Tours, France.
authors.forename: D
authors.initials: D
authors.lastname: Leroux
authors.forename: S
authors.initials: S
authors.lastname: Canépa
authors.forename: C
authors.initials: C
authors.lastname: Viskov
authors.forename: P
authors.initials: P
authors.lastname: Mourier
authors.forename: F
authors.initials: F
authors.lastname: Herman
authors.forename: J
authors.initials: J
authors.lastname: Rollin
authors.forename: Y
authors.initials: Y
authors.lastname: Gruel
authors.forename: C
authors.initials: C
authors.lastname: Pouplard
mesh_terms: D000906:Antibodies / Q000378:metabolism*; D000925:Anticoagulants / Q000009:adverse effects* / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D001666:Binding Sites, Antibody; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D016022:Case-Control Studies; D017984:Enoxaparin / Q000009:adverse effects* / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D006801:Humans; D015394:Molecular Structure; D015539:Platelet Activation / Q000187:drug effects*; D010978:Platelet Factor 4 / Q000276:immunology / Q000378:metabolism*; D018570:Risk Assessment; D012307:Risk Factors; D012701:Serotonin / Q000378:metabolism*; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D013921:Thrombocytopenia / Q000139:chemically induced* / Q000276:immunology
chemical_list: D000906:Antibodies; D000925:Anticoagulants; D017984:Enoxaparin; D012701:Serotonin; D010978:Platelet Factor 4