Heparin and homogeneous model heparin oligosaccharides form distinct complexes with protamine: Light scattering and zeta potential analysis

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 (300-700 kDa) 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.