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Enhancement of the epitopic activity of anti-thrombotic peptidomimetics by conjucation to a macromolecular carrier Zhang, Jerry Gongdu

Abstract

Background: Novel peptides with epitopic activity can be discovered by a combination of peptide arrays and computational interpolation. These peptides can mimic the activity of cellular receptors or their ligands, and are thus called peptidomimetics. The activity of peptidomimetics evidently varies in accordance with the attributes of the peptide. The fibrin-mimetic, Arginine-Glycine-Aspartate (RGD), is a well known anti-thrombotic peptide which inhibits the interaction between the platelet integrin GPITbIIIa and fibrinogenlfibrin. Short peptides often suffer from high inhibitory concentrations in vitro and short clearance times in vivo. To increase vascular residence times of such antithrombotic peptides, they were coupled to macromolecular carriers. Hyperbranched polyglycerols (I{PG), macromolecules designed as a dendritic carrier species, at a range of HPG molecular weights (MW) were tested as the carriers for antithrombotic peptidomimetics. Methods: HPG of MW 3 to 500 kDa were conjugated with RGD at a range of substitution ratios. The optimum molecular weight and substitution ratio were then applied to the peptide SHAYIGLKDR, a vWf mimic peptide discovered through the use of bioinformatics. For peptidomimetics (RGD and SHAYIGLKDR), enzymatic proteolysis was employed to distinguish the specificity of the inhibitory activity among HPG, peptide, and the HPG-peptide conjugate. Flow cytometry, UV spectroscopy, compound light microscopy, and lummiaggregometry were used to characterize the function of the conjugates in vitro. Results: Conjugation of RGD to HPG resulted in a decrease of the inhibitory concentration required to interrupt platelet-fibrinogen interactions by up to three orders of magnitude. Inhibitory activity was directly related to the number of peptides attached per HPG. Similar results were found when high molecular weight HPG, selected from the RGD-related experiments, was used to carry the peptide SHAYIGLKDR. None of HPG, RGD, SHAYIGLKDR, or their conjugates caused spontaneous platelet activation, or inhibited thrombin-mediated platelet activation, showing that the peptides’ activity is directed specifically toward their targets: GPllblllalfibrinogen (RGD) and GPIb/vWf (SHAYIGLKDR) interactions. Tryptic digestion of conjugates confirmed that the inhibitory activity of HPG conjugates was dependent on the presence of the intact peptides. Conclusions: Conjugation of peptidomimetics or other molecules to macromolecular platforms such as HPG is a viable method to enhance the peptidomimetics’ activity. The degree of enhancement is dependent upon the level of peptide substitution as well as the size of the carrier.

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