Heparin use in clinical settings is plagued by the polymer's heterogeneity, leading to challenges associated with production, purification, dosage, and antagonism when immediate reversal is needed.  To address these issues we have employed bacteriophage Q� nanoparticles, whose multi-valent nature makes it an attractive candidate for perturbing the coagulation mechanism that heparin polymers participate in.  First, we have developed antagonists that safely reverse the anti-coagulant activity of heparin.  To interact with the heparin polyanion, polycationic particles were generated both chemically and by mutation; in particular, mutant T18R, which mutates a single site on each of the 180 coat proteins that make up the capsid, showed full reversal of heparin activity.  Chromatography using a heparin-sepharose column confirmed a strong interaction between heparin and the T18R particle.  Binding studies using fluorescein-labeled heparin (HepFL) resulted in a concentration-dependent change in fluorescence intensity, which could be perturbed by the addition of unlabeled heparin.  Analysis of the fluorescence data yielded a dissociation constant of approximately 1 nM and a 1:1 binding stoichiometry for HepFL:VLP.  Studies using dynamic light scattering (DLS) suggest that T18R forms discrete complexes with heparin when the VLP:heparin molar ratios are equivalent.  In vitro clotting assays confirm the 1:1 binding stoichiometry as full antagonism of heparin is achieved when the particle to heparin molar ratio approaches unity.  These studies validate T18R for potential clinical use as an effective, non-toxic heparin antagonist.                We have also conducted studies towards the opposite approach:  generating specifically sulfated phage that may elicit heparin-like activity.  Surface lysine residues on Q� have been positioned to allow for maximum solvent accessibility; these positions are then modified with either singly or triply sulfated molecules.  Factor Xa and activated partial thrombin time assays are used to determine the efficacy of the sulfated nanoparticles in prolonging coagulation.

Virus nanoparticles that perturb the heparin-coagulation mechanism

247th National Meeting (2014)

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