title: Heparin and homogeneous model heparin oligosaccharides form distinct complexes with protamine: Light scattering and zeta potential analysis.
abstract: Large multimolecular complexes of heparin with positively charged proteins such as platelet factor 4 (PF4) or protamine can initiate immune responses associated with heparin use in patients, including the most significant adverse event, heparin-induced thrombocytopenia (HIT). Current evidence suggests that platelet-activating antibodies that recognize large multi-molecular complexes (300-700kDa) of PF4 bound to heparin cause HIT [1] and in very rare cases anti-protamine-heparin antibodies can induce thrombocytopenia [2]. Heparin is administered as a mixture of sulfated glycosaminoglycans of variable lengths and sulfation levels. To date the potential impact of chain length, sulfation level and impurities on the formation, size and immunogenicity of heparin-protamine complexes has not been addressed due to the lack of purified, homogenous heparin chains for testing purposes. Here, a set of well-characterized model heparin oligosaccharides was used with protamine sulfate to evaluate the physicochemical properties of the resulting complexes. Hydrodynamic radii and zeta potential profiles of heparin-protamine complexes were observed to be dependent upon the sulfation location, size and concentration of the model heparin oligosaccharides. The well-characterized oligosaccharide-protamine complexes analyzed in this work will be useful for establishing links between heparin-protamine complex physiochemical attributes to their potential to illicit cellular immunogenicity.
authors.affiliation: Food and Drug Administration, CDER, Division of Pharmaceutical Analysis, 63110 St Louis, MO, USA. Electronic address: Cynthia.Sommers@fda.hhs.gov.
authors.forename: Cynthia D
authors.initials: CD
authors.lastname: Sommers
authors.affiliation: Food and Drug Administration, CDER, Division of Pharmaceutical Analysis, 63110 St Louis, MO, USA.
authors.forename: Hongping
authors.initials: H
authors.lastname: Ye
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina, 27599 Chapel Hill, NC, USA.
authors.forename: Jian
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Linhardt
authors.affiliation: Food and Drug Administration, CDER, Division of Pharmaceutical Analysis, 63110 St Louis, MO, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Keire
mesh_terms: D006493:Heparin / Q000032:analysis*; D006801:Humans; D009844:Oligosaccharides; D010978:Platelet Factor 4; D011479:Protamines; D013921:Thrombocytopenia
chemical_list: D009844:Oligosaccharides; D011479:Protamines; D010978:Platelet Factor 4; D006493:Heparin
keywords: Complex; Heparin; Heparin induced thrombocytopenia (HIT); Protamine; Zeta (ξ) potential; dynamic light scattering