Heparin Antagonism by Polyvalent Display of Cationic Motifs on Virus‐Like Particles

Abstract

Polyvalent interactions allow biological structures to exploit low-affinity ligand–receptor binding events to affect physiological responses. We describe here the use of bacteriophage Qβ as a multivalent platform for the display of polycationic motifs that act as heparin antagonists. Point mutations to the coat protein allowed us to generate capsids bearing the K16M, T18R, N10R, or D14R mutations; because 180 coat proteins form the capsid, the mutants provide a spectrum of particles differing in surface charge by as much as +540 units (K16M vs. D14R). Whereas larger poly-Arg insertions (for example, C-terminal Arg8) did not yield intact virions, it was possible to append chemically synthesized oligo-Arg peptides to stable wild-type (WT) and K16M platforms. Heparin antagonism by the particles was evaluated by using the activated partial thrombin time (aPTT) clotting assay; this revealed that T18R, D14R, and WT-(R8G2)95 were the most effective at disrupting heparin-mediated anticoagulation (>95 % inhibition). This activity agreed with measurements of ζ potential (ZP) and retention time on cation exchange chromatography for the genetic constructs, which distribute their added positive charge over the capsid surface (+180 and +360 for T18R and D14R relative to WT). The potent activity of WT-(R8G2)95, despite its relatively diminished overall surface charge is likely a consequence of the particle’s presentation of locally concentrated regions with high positive charge density that interact with heparin’s extensively sulfated domains. The engineered cationic capsids retained their ability to inhibit heparin at high concentrations and showed no anticlotting activity of the kind that limits the utility of antiheparin polycationic agents that are currently in clinical use.

Engineered virus-like nanoparticles reverse heparin anticoagulation more consistently than protamine in plasma from heparin-treated patients

“…Virus-like particles (VLPs) derived from bacteriophage Qβ coat protein containing mutations of Thr18 to Arg (T18R) and of Lys16 to Met (K16M) were produced and isolated as previously described [15,16]. Purified particles were stored in 0.1 M HEPES, pH 7.4.…”

“…The Qβ virus-like particles (VLPs) are 28 nm in diameter. Through molecular biological and chemical manipulation we engineered polyvalent displays of cationic motifs on this scaffold, producing VLPs that bound to and blocked the anticoagulant function of UFH in biochemical assays [15]. Here we investigate the clinical heparin reversal function of one of these active VLPs, in which the solvent-exposed residue Thr18 of the coat protein was mutated to Arg (T18R), thereby giving the particle substantially greater positive surface charge.…”

“…Here we investigate the clinical heparin reversal function of one of these active VLPs, in which the solvent-exposed residue Thr18 of the coat protein was mutated to Arg (T18R), thereby giving the particle substantially greater positive surface charge. The K16M mutant serves as a negative control since its positive charge is diminished relative to the wild-type structure and it did not bind or reverse heparin in vitro [15]. …”

Targeting heparin and heparan sulfate protein interactions

“…Arginine-rich polycationic virus-like particles neutralize the heparin-antithrombin interaction in human plasma, 93 branched dendrimer-based peptides inhibited HSV-1 and 2 infection, 94 and a large peptide isolated from human hemofiltrate blocked HCMV infection in vitro through a HS-dependent pathway. 95 Synthetic ligands (e.g., 11 ) that form micelles can bind heparin through electrostatic interactions of their protonated amines (Figure 6).…”

Chemically Orthogonal Three‐Patch Microparticles

“…[3] While the spatially controlled presentation of chemical and biological ligands is well established for two-dimensional substrates, [4] very few methodologies exist for the spatially controlled decoration of three-dimensional objects, such as microparticles. [2f,5] Many of the patterning methods for two-dimensional substrates including photolithography, microcontact printing, dip-pen nanolithography, or blockcopolymer micelle nanolithography, [4d,6] are not easily extendable to the three-dimensional surfaces of microparticles.…”