We have reported that platelets can interact specifically with infected RBCs (iRBCs) and kill Plasmodium (1). Platelets release an anti-plasmodial agent called platelet factor 4 (PF4), a cytotoxic host-defence protein that enters iRBCs via interaction with erythrocyte Duffy antigen chemokine receptor (DARC) (2), where it then disrupts the parasite digestive vacuole (DV) membrane (3). Despite this desirable activity, PF4 is a poor antimalarial drug lead because it has a chemokine sequence that induces immunotoxic side-effects at high doses (4), and its activity is dependent on DARC, and so is ineffective in people with suppressed DARC expression. Early attempts to remove the undesired chemokine and DARC-dependent functions, while retaining anti-plasmodial activity of PF4, resulted in peptides with poor potency (3).
Using a new approach, we engineered a novel peptide containing the isolated anti-plasmodial domain of PF4, which through cyclization retained the critical structure of the parent protein. The peptide, called cPF4PD (cyclic PF4 peptide dimer) has an anti-plasmodial potency similar to PF4, but it lacks chemokine functions and does not require DARC. A comprehensive characterisation of the parasite killing mechanism revealed several unique biophysical and biological properties, including: i) stability in human serum; ii) selective targeting and accumulation within iRBCs; iii) ability to cross host and parasite cell membranes without lysis; iv) killing of intracellular parasites by disruption of the DV membrane. This selective and completely novel activity against Plasmodium was accounted by observations of the peptide’s specific binding to and penetration of membranes with exposed negatively-charged phospholipid headgroups.
As well as demonstrating a promising example of a novel anti-plasmodial molecule based on a human host defence protein, our findings indicate the potential of cPF4PD as a novel cell-penetrating scaffold for delivering bioactive cargoes to inhibit intracellular Plasmodium-specific proteins critical for parasite survival.