GPVI and GPIbα Mediate Staphylococcal Superantigen-Like Protein 5 (SSL5) Induced Platelet Activation and Direct toward Glycans as Potential Inhibitors

Baker IDI Heart & Diabetes Institute, Melbourne, Victoria, Australia.
PLoS ONE (Impact Factor: 3.53). 04/2011; 6(4):e19190. DOI: 10.1371/journal.pone.0019190
Source: PubMed

ABSTRACT 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.
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 GPIbα 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.
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.

Download full-text


Available from: Ingo Ahrens, Jun 19, 2015
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The photoinitiated radical reactions between thiols and alkenes/alkynes (thiol-ene and thiol-yne chemistry) have been applied to a functionalization methodology to produce carbohydrate-presenting surfaces for analyses of biomolecular interactions. Polymer-coated quartz surfaces were functionalized with alkenes or alkynes in a straightforward photochemical procedure utilizing perfluorophenylazide (PFPA) chemistry. The alkene/alkyne surfaces were subsequently allowed to react with carbohydrate thiols in water under UV-irradiation. The reaction can be carried out in a drop of water directly on the surface without photoinitiator, and any disulfide side products were easily washed away after the functionalization process. The resulting carbohydrate-presenting surfaces were evaluated in real-time studies of protein-carbohydrate interactions using a quartz crystal microbalance (QCM) flow-through system with recurring injections of selected lectins, with intermediate regeneration steps using low pH buffer. The resulting methodology proved fast, efficient and scalable to high-throughput analysis formats, and the produced surfaces showed significant protein binding with expected selectivities of the lectins used in the study.
    Biosensors & Bioelectronics 01/2012; 34(1):51-6. DOI:10.1016/j.bios.2012.01.001 · 6.45 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Nucleotide-based drug candidates such as antisense oligonucleotides, aptamers, immunoreceptor-activating nucleotides, or (anti)microRNAs hold great therapeutic promise for many human diseases. Phosphorothioate (PS) backbone modification of nucleotide-based drugs is common practice to protect these promising drug candidates from rapid degradation by plasma and intracellular nucleases. Effects of the changes in physicochemical properties associated with PS modification on platelets have not been elucidated so far. Here we report the unexpected binding of PS-modified oligonucleotides to platelets eliciting strong platelet activation, signaling, reactive oxygen species generation, adhesion, spreading, aggregation, and thrombus formation in vitro and in vivo. Mechanistically, the platelet-specific receptor glycoprotein VI (GPVI) mediates these platelet-activating effects. Notably, platelets from GPVI function-deficient patients do not exhibit binding of PS-modified oligonucleotides, and platelet activation is fully abolished. Our data demonstrate a novel, unexpected, PS backbone-dependent, platelet-activating effect of nucleotide-based drug candidates mediated by GPVI. This unforeseen effect should be considered in the ongoing development programs for the broad range of upcoming and promising DNA/RNA therapeutics. © 2015 Flierl et al.
    Journal of Experimental Medicine 02/2015; 212(2). DOI:10.1084/jem.20140391 · 13.91 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: New methods for analysing both platelet and plasma forms of the platelet-specific collagen receptor, glycoprotein VI (GPVI) in experimental models or human clinical samples, and the development of the first therapeutic compounds based on dimeric soluble GPVI-Fc or anti-GPVI antibody-based constructs, coincide with increased understanding of the potential pathophysiological role of GPVI ligand binding and shedding. Platelet GPVI not only mediates platelet activation at the site of vascular injury where collagen is exposed, but is also implicated in the pathogenesis of other diseases, such as atherosclerosis and coagulopathy, rheumatoid arthritis and tumour metastasis. Here, we describe some of the critical mechanisms for generating soluble GPVI from platelets, and future avenues for exploiting this unique platelet-specific receptor for diagnosis and/or disease prevention.
    Thrombosis and Haemostasis 01/2012; 107(4):648-55. DOI:10.1160/TH11-10-0745 · 5.76 Impact Factor