Although they are remarkably diverse in function, G-protein-coupled receptors (GPCRs) share a highly conserved topology of a seven-transmembrane helical core domain joined by three intracellular loops and three extracellular loops. In the October issue of Nature Medicine, Covic et al. showed that the attachment of a palmitate group to peptide sequences derived from the third intracellular loop of the GPCRs protease-activated receptor 1 (PAR1) and PAR4 resulted in the production of potent antagonists of receptor signalling. These cell-penetrating lipidated peptides, known as pepducins, can be used as tools to delineate the physiological roles of GPCRs and determine the therapeutic value of blockade of a particular signalling pathway.

The large number of GPCRs and their role in disease have made them attractive drug targets. As most GPCR agonists and antagonists act on the extracellular surface of the receptor, the intracellular surface has not been exploited for therapeutic targets. However, pepducins act as receptor-modulating agents by targeting the intracellular surface of the GPCR.

PAR1 and PAR4 are activated by the protease thrombin, which results in the activation of platelets. The ability to control this signalling cascade would be useful in preventing thrombotic complications associated with heart attacks and stroke. The present study set out to develop and test PAR1- and PAR4-based pepducin antagonists for their ability to block platelet activation and thrombosis in vivo.

Soluble peptide ligands of PAR1 and PAR4 were inhibited by their respective pepducins. Pretreatment of human platelets with anti-PAR1 pepducin completely blocked aggregation in response to 3 nM thrombin, a physiological concentration. Aggregation was not blocked in response to 20 nM thrombin, presumably because of the signal from PAR4. However, the anti-PAR4 pepducin inhibited most aggregation in response to 20 nM thrombin, indicating that the anti-PAR4 pepducin also inhibits PAR1 signalling. Infusion of the anti-PAR4 pepducin into mice extended bleeding time and protected against systemic platelet activation, consistent with the phenotype of a Par4-deficient mouse. As expected, the anti-PAR1 pepducin did not have any effect on bleeding time, as mouse platelets lack Par1.

The use of pepducins provides a powerful and general approach to determine the effect of disruption of GPCR signalling. This will be particularly useful for GPCRs for which there are no known extracellular antagonists, or in cases in which genetic deficiency results in embryonic death. In addition, the pepducins themselves could be used as therapeutics. In fact, unlike many small-peptide drugs, such as the platelet-receptor inhibitor eptifibatide, these lipidated peptides have long half-lives. This is probably because they partition to cell membranes, where they become resistant to clearance and attack by proteases.