PB0775 - Disruption of the interaction of platelets with leukocytes by a novel short synthetic peptide targeting P-selectin

Background: The interaction of platelets and leukocytes plays an important role in inflammation and thrombosis. The discovery of novel pharmacological tools able to reduce the formation of platelet-leukocyte complexes is therefore a promising yet unexplored aspect of cardiovascular research.

Aims: Based on previous studies showing that the N-terminal domain of S. aureus extracellular fibrinogen-binding protein (Efb) binds to P-selectin and interferes with platelet-leukocyte aggregate formation, here we aimed to identify the minimal Efb motif required for binding platelets and to investigate its effect on the interaction of platelets with leukocyte.

Methods: Using a library of synthetic peptides, we mapped the platelet-binding region of the N-terminal domain of Efb using dot blots, pull-down assays and flow cytometry. The effect of the peptide on platelets was tested by aggregometry, flow cytometry, immunoblotting, and in vitro thrombus formation assays. The effect on platelet-leukocyte interactions was tested by flow cytometry and cell imaging.

Results: Efb68-87 is a 20 amino acid-long peptide identified for its ability to bind to resting and, to a greater extent, thrombin-stimulated platelets in the absence of fibrinogen. Competitive binding experiments utilising P-selectin glycoprotein ligand-1 (PSGL-1) identified P-selectin as the cellular docking site mediating platelet binding of Efb68-87. Accordingly, Efb68-87 did not bind to other blood cells and captured platelets from human whole blood under low shear stress conditions. Efb68-87 did not affect platelet activation, but inhibited the formation of platelet-leukocyte aggregates (PLAs). In addition, Efb68-87 also abolished the platelet-dependent stimulation of neutrophil extracellular traps (NETs), which have been shown to contribute to the vascular complications of acute and chronic inflammation, and sepsis.

Conclusion(s): We have identified Efb68-87 as a peptide selectively binding P-selectin and inhibiting the interaction of platelets with leukocytes. Efb68-87 is therefore an exciting candidate for the development of novel selective antagonists of the proinflammatory activity of platelets.