Background: Staphylococcus aureus (S. aureus) is a common pathogen capable of causing life-threatening infections. Staphylococcal superantigen-like protein 5 (SSL5) has recently been shown to bind to platelet glycoproteins and induce platelet activation. This study investigates further the interaction between SSL5 and platelet glycoproteins. Moreover, using a glycan discovery approach, we aim to identify potential glycans to therapeutically target this interaction and prevent SSL5-induced effects. Methodology/Principal Findings: In addition to platelet activation experiments, flow cytometry, immunoprecipitation, surface plasmon resonance and a glycan binding array, were used to identify specific SSL5 binding regions and mediators. We independently confirm SSL5 to interact with platelets via GPIbI? and identify the sulphated-tyrosine residues as an important region for SSL5 binding. We also identify the novel direct interaction between SSL5 and the platelet collagen receptor GPVI. Together, these receptors offer one mechanistic explanation for the unique functional influences SSL5 exerts on platelets. A role for specific families of platelet glycans in mediating SSL5-platelet interactions was also discovered and used to identify and demonstrate effectiveness of potential glycan based inhibitors in vitro. Conclusions/Significance: These findings further elucidate the functional interactions between SSL5 and platelets, including the novel finding of a role for the GPVI receptor. We demonstrate efficacy of possible glycan-based approaches to inhibit the SSL5-induced platelet activation. Our data warrant further work to prove SSL5-platelet effects in vivo. A? 2011 Hu et al.