RGD-Substituted High Molecular Weight Hyper-Branched Polyglycerols (HPG) Are Effective Platelet Inhibitors.

Peptides containing Arg-Gly-Asp (RGD) sequences are known to bind to integrins which mediate cell adhesion and therefore have been utilized in applications such as antithrombotics and tissue engineering. Although RGD and related peptides show promise, their unfavourable pharmacokinetic profiles and susceptibility to in vivo proteolysis hinder their clinical usefulness. Peptide-polymer conjugates can address one of these challenges by extending the peptide’s residence in plasma. Hyperbranched polyglycerols (HPGs) are biocompatible polyether polyols with very long plasma circulation half-lives (t_1/2 ∼ 60h) and as such are ideally suited to be carriers in such conjugates. HPGs of three different molecular weights were conjugated with RGD peptides at various substitution levels. Some of the terminal hydroxyl groups of polyglycerols were converted to vinyl sulfone groups which were subsequently utilized to couple cysteine terminated RGD peptides. The following conjugates were made: 515 kDa with substitution levels of 100:1, 500:1 and 1000:1; 100 kDa with substitution levels of 100:1 and 1000:1; and 3 kDa with substitution levels of 1:1, and 10:1. RGD-coupled HPG inhibited fibrinogen binding to platelet glycoprotein IIb-IIIa as detected by flow cytometry using anti-fibrinogen. Compared to free RGD (I_c50 = 5 x 10^-5 M), inhibition of fibrinogen binding increased with increasing HPG molecular weight and increasing RGD-substitution for the high MW conjugates: I_c50 for the 515 kDa conjugates were 6.3 x 10⁻⁸ M for the 100:1 substitution, 4.7 x 10^-8 M for the 500:1 substitution, and 4.1 x 10⁻⁸ M for 1000:1 substitution; I_c50 for the 100 kDa conjugates were 7.0 x 10⁻⁷ M for the 100:1 substitution and 1.2 x 10⁻⁷ M for the 1000:1 substitution. I_c50 for the 3 kDa conjugates were 2 x 10⁻⁵ M for the 1:1 substitution, 2 x 10⁻⁴ M for 10:1 substitution. Similarly, platelet function, as demonstrated by MnCl₂-initated aggregation was inhibited in a dose- and molecular weight-dependent manner by HPG-RGD conjugates. Platelet aggregate formation and aggregate size were confirmed by microscopy. However, unsubstituted HPG had no effect on platelet fibrinogen binding and neither conjugated nor unconjugated HPG increased platelet CD62 surface expression. Trypsin digestion (but not SBTI-treated trypsin) removed the inhibitory activity of the conjugates and thus confirmed that both fibrinogen binding and aggregation-inhibition were dependent on the RGD peptide sequence. Such multi-determinant peptide-HPG conjugates represent a new approach to the development of antithrombotic drugs.