title: Directed polyvalent display of sulfated ligands on virus nanoparticles elicits heparin-like anticoagulant activity.
abstract: Heparin is a sulfated glycosaminoglycan that is widely used as an anticoagulant. It is typically extracted from porcine or bovine sources to yield a heterogeneous mixture that varies both in molecular weight and in degree of sulfation. This heterogeneity, coupled with concern for contamination, has led to widespread interest in developing safer alternatives. Described herein are sulfated bacteriophage Qβ virus-like particles (VLPs) that elicit heparin-like anticoagulant activity. Sulfate groups were appended to the VLP by synthesis of single- and triple-sulfated ligands that also contained azide groups. Following conversion of VLP surface lysine groups to alkynes, the sulfated ligands were attached to the VLP via copper-catalyzed azide-alkyne cycloaddition (CuAAC). MALDI-MS analysis of the intermediate alkyne VLP indicated that the majority of the coat proteins contained 5-7 of the alkyne linkers; similar analysis of the intermediate alkyne particles conjugated to a fluorescein azide suggest that nearly the same number of attachment points (3-6) are modified via CuAAC. Analysis by SDS-PAGE with fluorescent staining indicated altered migration patterns for the various constructs: compared to the wild-type nanoparticle, the modified coat proteins appeared to migrate farther toward the positive pole in the gel, with coat proteins displaying the triple-sulfated ligand migrating significantly farther. Clotting activity analyzed by activated partial thrombin time (APTT) assay showed that the sulfated particles were able to perturb coagulation, with VLPs displaying the triple-sulfated ligand approximately as effective as heparin on a per mole basis; this activity could be partially reversed by protamine. ELISA experiments to assess the response of the complement system to the VLPs indicate that sulfating the particles may reduce complement activation.
authors: Department of Chemistry, Occidental College , Los Angeles, California 90041, United States.
authors: Griffin
authors: G
authors: Mead
authors: Megan
authors: M
authors: Hiley
authors: Taryn
authors: T
authors: Ng
authors: Conrad
authors: C
authors: Fihn
authors: Kevin
authors: K
authors: Hong
authors: Myles
authors: M
authors: Groner
authors: Walker
authors: W
authors: Miner
authors: Daniel
authors: D
authors: Drugan
authors: William
authors: W
authors: Hollingsworth
authors: Andrew K
authors: AK
authors: Udit
mesh_terms: D000480:Alkynes / Q000737:chemistry; D017908:Allolevivirus / Q000737:chemistry*; D000925:Anticoagulants / Q000737:chemistry* / Q000494:pharmacology*; D001386:Azides / Q000737:chemistry; D001777:Blood Coagulation / Q000187:drug effects; D036022:Capsid Proteins / Q000737:chemistry; D003167:Complement Activation / Q000187:drug effects; D003300:Copper / Q000737:chemistry; D061565:Cycloaddition Reaction; D006493:Heparin / Q000737:chemistry* / Q000494:pharmacology*; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D053758:Nanoparticles / Q000737:chemistry*; D010314:Partial Thromboplastin Time; D011487:Protein Conformation; D013431:Sulfates / Q000737:chemistry*
chemical_list: D000480:Alkynes; D000925:Anticoagulants; D001386:Azides; D036022:Capsid Proteins; D008024:Ligands; D013431:Sulfates; D003300:Copper; D006493:Heparin