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: 3.23). 04/2011; 6(4):e19190. DOI: 10.1371/journal.pone.0019190
Source: PubMed


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,
113 Reads
  • Source
    • "(Kiessling and Splain 2010; Lepenies and Seeberger 2010; Li and Richards 2010; Seeberger 2009; Varki et al. 2009; Wu and Wong 2011) For example, different carbohydrate array methodologies have recently been designed and evaluated for high-throughput analysis of protein-carbohydrate interactions. (Horlacher and Seeberger 2008; Krishnamoorthy and Mahal 2009; Lee and Shin 2005; Oyelaran and Gildersleeve 2009; Park et al. 2008; Park and Shin 2007; Pei et al. 2007c; Tyagi et al. 2010; Wu et al. 2009) Glycan arrays have thus been used to identify proteins involved in cancer metastasis, (Hatakeyama et al. 2009) enzymes involved in wound healing, (Saravanan et al. 2010) and glycans modulating T cell death; (Earl et al. 2010) to evaluate blood serum glycan binding, (Huflejt et al. 2009) antibodies towards HIV, (Luallen et al. 2010) and antibodies for use in cancer treatment; (Huang et al. 2006; Nagre et al. 2010; Sawada et al. 2011) to evaluate the binding specificity of glycan-binding proteins and receptors; (Feinberg et al. 2010; Gout et al. 2010; Hoorelbeke et al. 2011; Horlacher et al. 2011; Pipirou et al. 2011; Porter et al. 2010; Singh et al. 2009) to investigate the binding specificities of disease causing bacteria, (Hu et al. 2011) viruses, (Krishnamoorthy et al. 2009; Neu et al. 2010; Nilsson et al. 2011) and fungi;(Chachadi et al. 2011) as well as for the in-depth investigation of avian and swine influenza viruses.(de Vries et al. 2011; Lao et al. 2011; Liao et al. 2010; Pappas et al. 2010; Stevens et al. 2010; Xu et al. 2010) Although such glycan array methodologies yield significant knowledge of glycan interactions, there are still obstacles to overcome in the production of universally valid array methodologies. "
    [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 · 6.41 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 · 4.98 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Staphylococcus aureus can induce platelet aggregation. The rapidity and degree of this correlates with the severity of disseminated intravascular coagulation, and depends on platelet peptidoglycans. Surface-located thiol isomerases play an important role in platelet activation. The staphylococcal extracellular adherence protein (Eap) functions as an adhesin for host plasma proteins. Therefore we tested the effect of Eap on platelets. We found a strong stimulation of the platelet-surface thiol isomerases protein disulfide isomerase and endoplasmic reticulum stress proteins 57 and 72 by Eap. Eap induced thiol isomerase-dependent glycoprotein IIb/IIIa activation, granule secretion, and platelet aggregation. Treatment of platelets with thiol blockers, bacitracin, and anti-protein disulfide isomerase antibody inhibited Eap-induced platelet activation. The effect of Eap on platelets and protein disulfide isomerase activity was completely blocked by glycosaminoglycans. Inhibition by the hydrophobic probe bis(1-anilinonaphthalene 8-sulfonate) suggested the involvement of hydrophobic sites in protein disulfide isomerase and platelet activation by Eap. In the present study, we found an additional and yet unknown mechanism of platelet activation by a bacterial adhesin, involving stimulation of thiol isomerases. The thiol isomerase stimulatory and prothrombotic features of a microbial secreted protein are probably not restricted to S aureus and Eap. Because many microorganisms are coated with amyloidogenic proteins, it is likely that the observed mechanism is a more general one.
    Arteriosclerosis Thrombosis and Vascular Biology 04/2012; 32(8):1979-90. DOI:10.1161/ATVBAHA.112.246249 · 6.00 Impact Factor
Show more