title: Heparin modifies the immunogenicity of positively charged proteins.
abstract: The immune response in heparin-induced thrombocytopenia is initiated by and directed to large multimolecular complexes of platelet factor 4 (PF4) and heparin (H). We have previously shown that PF4:H multimolecular complexes assemble through electrostatic interactions and, once formed, are highly immunogenic in vivo. Based on these observations, we hypothesized that other positively charged proteins would exhibit similar biologic interactions with H. To test this hypothesis, we selected 2 unrelated positively charged proteins, protamine (PRT) and lysozyme, and studied H-dependent interactions using in vitro and in vivo techniques. Our studies indicate that PRT/H and lysozyme/H, like PF4/H, show H-dependent binding over a range of H concentrations and that formation of complexes occurs at distinct stoichiometric ratios. We show that protein/H complexes are capable of eliciting high-titer antigen-specific antibodies in a murine immunization model and that PRT/H antibodies occur in patients undergoing cardiopulmonary bypass surgery. Finally, our studies indicate that protein/H complexes, but not uncomplexed protein, directly activate dendritic cells in vitro leading to interleukin-12 release. Taken together, these studies indicate that H significantly alters the biophysical and biologic properties of positively charged compounds through formation of multimolecular complexes that lead to dendritic cell activation and trigger immune responses in vivo.
authors.affiliation: Division of Hematology, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: Shalini L
authors.initials: SL
authors.lastname: Chudasama
authors.forename: Benjamin
authors.initials: B
authors.lastname: Espinasse
authors.forename: Fred
authors.initials: F
authors.lastname: Hwang
authors.forename: Rui
authors.initials: R
authors.lastname: Qi
authors.forename: Manali
authors.initials: M
authors.lastname: Joglekar
authors.forename: Galyna
authors.initials: G
authors.lastname: Afonina
authors.forename: Mark R
authors.initials: MR
authors.lastname: Wiesner
authors.forename: Ian J
authors.initials: IJ
authors.lastname: Welsby
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Ortel
authors.forename: Gowthami M
authors.initials: GM
authors.lastname: Arepally
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology*; D001323:Autoantibodies / Q000276:immunology*; D002315:Cardiopulmonary Bypass; D003713:Dendritic Cells / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D007114:Immunization; D051379:Mice; D008810:Mice, Inbred C57BL; D009113:Muramidase / Q000276:immunology* / Q000378:metabolism; D010978:Platelet Factor 4 / Q000276:immunology / Q000378:metabolism; D011479:Protamines / Q000276:immunology* / Q000378:metabolism
chemical_list: D000925:Anticoagulants; D001323:Autoantibodies; C082073:PRM1 protein, human; C513176:Prm1 protein, mouse; D011479:Protamines; D010978:Platelet Factor 4; D006493:Heparin; D009113:Muramidase