| Summary: | 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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