GPVI and GPIba Mediate Staphylococcal Superantigen-
Like Protein 5 (SSL5) Induced Platelet Activation and
Direct toward Glycans as Potential Inhibitors
Houyuan Hu1,2., Paul C. J. Armstrong1., Elie Khalil1, Yung-Chih Chen1,3, Andreas Straub1, Min Li1,2,
Juliana Soosairajah1, Christoph E. Hagemeyer1, Nicole Bassler1, Dexing Huang1, Ingo Ahrens1, Guy
Krippner1, Elizabeth Gardiner4, Karlheinz Peter1,3*
1Baker IDI Heart & Diabetes Institute, Melbourne, Victoria, Australia, 2Department of Cardiology, Southwest Hospital, Third Military Medical University, Chongqing, China,
3Department of Medicine, Monash University, Melbourne, Victoria, Australia, 4Australian Centre for Blood Diseases, Monash University, Melbourne, Victoria, Australia
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
Methodology/Principal Findings: In addition to platelet activation experiments, flow cytometry, immunoprecipita-
tion, 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 GPIba 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
Citation: Hu H, Armstrong PCJ, Khalil E, Chen Y-C, Straub A, et al. (2011) GPVI and GPIba Mediate Staphylococcal Superantigen-Like Protein 5 (SSL5) Induced
Platelet Activation and Direct toward Glycans as Potential Inhibitors. PLoS ONE 6(4): e19190. doi:10.1371/journal.pone.0019190
Editor: Herman Tse, The University of Hong Kong, Hong Kong
Received December 16, 2010; Accepted March 22, 2011; Published April 28, 2011
Copyright: ? 2011 Hu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was funded by the National Health & Medical Research Council (NHMRC) of Australia. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Staphylococcus aureus (S. aureus) is one of the most common and
dangerous bacterial pathogens to humans. It can cause a range of
diseases including life-threatening thromboembolic diseases such
as infective endocarditis [1,2] and disseminated intravascular
coagulation (DIC) . S. aureus is increasingly found in the
community and hospitals, and is one of the most common
bacterium isolated from blood cultures. Additionally, its growing
resistance to multiple drugs means this pathogen presents a major
clinical challenge .
The interaction of S. aureus, via a variety of surface-associated
proteins with platelets has recently been recognized to play a
significant pathophysiological role in S. aureus-associated diseases
. These direct interactions between S. aureus and platelets result
in bacteria-platelet aggregates which, for example, are charac-
teristic of S. aureus endocarditis . S. aureus is known to secrete
the Staphylococcal superantigen-like proteins (SSLs) that are
structurally homologous to the superantigens (SAg), but do not
seem to exhibit the same functions . The SSLs are a group of
related genes which are all clustered on a genomic island [8,9,10].
This pathogenicity island contains between 7 and 11 SSL genes
[9,11] and is present in all strains of S. aureus examined to date
While SSLs have previously been implicated in S. aureus
virulence , recent studies showed that two of these toxins,
SSL5 and SSL11, can bind to P-selectin glycoprotein ligand-1
(PSGL-1) on granulocytes to inhibit P-selectin-mediated neutro-
phil rolling and the subsequent migration of neutrophils to sites
of infection [7,13,14], and inhibit leukocyte activation by
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chemokines and anaphylatoxins . PSGL-1 is structurally and
functionally related to the GPIba subunit of the platelet GPIb-
IX-V receptor complex. Indeed, SSL5 has recently been
demonstrated to cause platelet activation, associated with
interactions between SSL5 and either GPIba or GPIIb/IIIa
. Furthermore, as these receptors are membrane-associated
sialomucins containing large clusters of O-linked sugar chains
and have been shown to bind P-selectin [17,18,19,20], a role for
glycans has been implicated .
In this study, we independently corroborate that SSL5 can
induce in vitro platelet activation and also possesses the ability of
binding to platelet membrane receptor GPIba, with the
sulphated-tyrosine residues playing a significant role. We also
identify the novel interaction between SSL5 and the collagen
receptor GPVI . Furthermore, our study defines specific
glycan families which are important in mediating the SSL5-
platelet interaction, and demonstrate potential glycan based
therapeutic approaches to inhibit SSL5-induced platelet activa-
SSL5 specifically binds to human platelets in a
Figure 1A shows purified recombinant SSL5migratingas a single
band of ,27 kDa.PurifiedSSL5 has been previously shown to bind
to the human leukemic HL60 monocytic cell line [13,22] and we
confirmed that our recombinant SSL5 protein, but not a non-
functional mutant form of SSL5 carrying a T175P point mutation
(T175P)  binds to HL60 cells by flow cytometry. In addition,
SSL5 blocked the binding of anti-PSGL-1 mAb KPL-1 to HL60
cells (data not shown). SSL5 binding to human washed platelets,
detected by flow cytometry, increased in a concentration-dependent
manner, contrasting with T175P which did not (figures 1B and 1C).
Figure 1. Analysis of purified recombinant SSL5 and flow cytometric examination of SSL5 binding to human platelets. (A) Purification
of a ,27 kDa SSL5 protein from BL21 E. coli. Samples were analyzed by SDS-PAGE and Coomassie blue staining and by Western blot using anti-His6
mAb as described in Supplemental Materials and Methods S1. (B) A representative flow cytometry histogram of platelets incubated with 10 mg/ml
SSL5 or SSL5 mutant T175P. (C) Washed platelets were incubated with 0.1–80 mg/ml of either SSL5 or T175P SSL5 followed by Alexa Fluor 488-
conjugated anti-penta-His mAb and fluorescence intensity was measured by flow cytometry. Data is expressed as geometric mean fluorescence
intensity 6 SEM of three independent experiments.
SSL5 Binding Platelets
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Activation and inhibition of platelets by SSL5
Surface levels of P-selectin (figure 2A), and the activation of
the integrin GPIIb/IIIa, using the active conformation-specific
mAb PAC-1 (figure 2B) were assessed as measures of platelet
activation. Treatment with SSL5 but not T175P induced
increased levels of P-selectin and active GPIIb/IIIa on platelets,
to levels comparable to those observed after P2Y- or GPVI-
dependent activation of platelets. Aggregation of human washed
platelets after treatment with SSL5, but not the mutant SSL5
(data notshown),was confirmed
aggregometry (figure 2C), and which was inhibited by prior
incubation (5 min) with either a spleen tyrosine kinase (BAY61-
3606, 5 mM)  or src (PP2, 3 mM)  inhibitor. SSL5-
treated platelets also firmly adhered and spread on a fibrinogen-
matrix under static conditions in a dose-dependent manner
(figure 3A and 3B).
by light transmission
Figure 2. SSL5 activates human platelets and induces syk and src dependent platelet aggregation. (A) Levels of anti-P-selectin mAb or
(B) PAC-1 binding were measured by flow cytometry in samples of washed platelets after incubation with PBS, 20 mM ADP, 100 ng/ml convulxin, or
30 mg/ml SSL5 or T175P SSL5. (C) Representative aggregation trace obtained in washed platelets, induced by 20 mg/ml SSL5 or no stimulation (PBS).
Aggregation was strongly inhibited by pre-treatment of platelet with either a syk kinase inhibitor (BAY61-3606, 5 mM), or a src inhibitor (PP2, 3 mM).
Images are representative of three independent experiments. * P,0.05.
SSL5 Binding Platelets
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Identification of SSL5-binding epitopes on GPIba
Using immunoprecipitation we confirmed the co-isolation of
SSL5 with PSGL-1 from HL60 cells using an anti-PSGL-1 mAb
(data not shown), consistent with previous reports of SSL5
characteristics [13,22]. Using anti-GPIba (WM23) or isotype
control (mouse IgG1), SSL5 was detected in GPIb-specific
immunoprecipitates but not isotype controls (figure 4A), confirm-
ing a molecular interaction between GPIba and SSL5. Direct
interaction between SSL5 and GPIba was confirmed by surface
plasmon resonance using recombinant fusion proteins containing
the N-terminal ligand-binding region fused to a C-terminal human
IgG domain (figure 4B). No interaction was seen when a control
human IgG domain alone was used or when the SSL5 mutant
T175P was used as the analyte (data not shown). Concentrations of
SSL5 above 80 nM were also assessed but found to display
inconsistent binding characteristics under the experimental
conditions used; possibly due to the cationic nature of the
Identification of binding sites on GPIba was done by pre-
incubating platelets with mAbs raised against separate epitopes of
GPIba, and the subsequent binding of SSL5 assessed using flow
cytometry. Binding of SSL5 (3 and 10 mg/ml) and platelets, was
significantly inhibited by the anti-GPIba mAb SZ2 (p,0.01),
identifying the sulphated-tyrosine region as a key binding site. The
mAbs AK2 (first leucine-rich repeat region) and WM23 (macro-
glycopeptide region) only weakly inhibited this interaction,
whereas the greatest level of inhibition was achieved by co-
incubating all three mAbs with platelets (p,0.001; figure 4C–G).
Interaction between SSL5 and GPVI
Preincubation of SSL5 with a recombinant ectodomain fragment
of GPVI, containingthe two immunoglobulin-like domains with the
ligand binding region but not the mucin-like domain, equivalent to
twice the concentration of SSL5 inhibited SSL5-induced, but not
ADP-induced, P-selectin expression by 75% (p,0.001; figure 5A).
A direct interaction was demonstrated using surface plasmon
resonance using a GPVI recombinant protein consisting of N-
terminal ligand-binding region (residues 21–234) fused to a C-
terminal human IgG domain (figure 5B). No interaction was found
between GPVI or human IgG domain coating when the T175P
analyte was used (data not shown). Concentrations of SSL5 above
80nM were also assessed but found to display inconsistent binding
characteristics under the experimental conditions used; possibly due
to the cationic nature of the molecule.
SSL5 glycan binding specificity and ability to inhibit SSL5
effects in vitro
Binding of SSL5 to platelets treated with neuraminidase, to
remove sialic acid from the platelet surface, was almost completely
lost (figure 6A), indicating a role for sialic acid residues in platelet-
SSL5 interactions. A glycan microarray was undertaken and
screened 377 different glycans for binding ability. The strongest
20 binders are presented in Table 1. Notably, the trisaccharide
sialyllactosamine (sLacNac - Neu5Aca2-3Galb1-4GlcNAc) termi-
nus was present in all but one (entry 12) of the high affinity glycans,
and the PSGL-1 tetrasaccharide sialyl Lewis X (sLeX: Neu5Aca2-
3Galb1-4(Fuca1-3)GlcNAc) was well represented in the glycans
with highest affinity. Additionally, SSL5 was found to bind to eight
of theten strongestSSL11-binding glycans. Using recombinant
SSL5 we investigated the use of glycans as potential drug candidates
for SSL5 blocking properties. Flow cytometric analysis showed that
incubation of each of the glycans sLeX, sLacNac, sialic acid
glycoside, at a final concentration of 100 mM both reduced the
levels of SSL5 binding (figure 6B) and inhibited the platelet-
activating effect of SSL5. The sLacNac and sialic acid glycoside
glycans were able to inhibit SSL5 (10 mg/ml)-induced platelet
activation by 86% (p,0.001) and 35% (p=0.059), respectively
(figure 6C). No effect was observed on ADP-induced activation
(data not shown). The glycan-based drug candidate bimosiamose,
inhibited both SSL5-induced P-selectin expression and GPIIb/IIIa
activation in a concentration-dependent manner between the
concentrations of 10 mM and 1 mM (figures 6D and 6E).
It has recently been shown that SSL5, an exotoxin secreted by S.
aureus, can interact with the platelet glycoproteins GPIba causing
platelet activation . Using flow cytometry, immunoprecipita-
tion, a glycan binding array and surface plasmon resonance, we
have independently confirmed GPIba as a receptor for SSL5 on
platelets and identified some of the molecular regions of GPIba
that support SSL5 binding to the platelet surface. Furthermore,
our data identifies GPVI as also potentially contributing to the
localization of SSL5 with the platelet surface.
Figure 3. SSL5 induces spreading of platelets on fibrinogen. Gel
filtered platelets (GFP) were incubated with 20 mM ADP, 1–10 mg/ml
SSL5, 10 mg/ml T175P (mutant SSL5) at 37uC for 30 minutes on
fibrinogen. (A) Representative pictures were obtained by DIC (660)
following adhesion. (B) Presented data are means 6 SEM of four
independent experiments using five separate fields per experiment.
** P,0.01, *** P,0.001 compared to control (PBS).
SSL5 Binding Platelets
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SSL5 Binding Platelets
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The recent publication by de Haas et al , did not specify the
precise SSL5-binding region on GPIba, although the authors reported
rich-repeat of GPIba, had little effect on SSL5 binding. In this study,
we used a range of epitope-specific GPIba antibodies to examine the
nature of SSL5/GPIba binding. We found that the SZ2 antibody,
which binds to the anionic sulphated-tyrosine region, had a significant
AK2, WM23 and SZ2 antibodies were used in combination, more
than 75% inhibition of SSL5 binding was observed. We report here a
PSGL-1-mediated binding of SSL5 to HL60 cells, and other members
of the SSL family have been reported to bind PSGL-1 which contains
an N-terminal sulfated-tyrosine sequence remarkably similar to the
anionic region of GPIba. P-selectin utilises these negatively charged
regions to bind to both PSGL-1 and GPIba. GPIba is a sialomucin
 and can bind ligands via sLex-related carbohydrates. Our finding
that the SSL5 mutant T175P, which could no longer bind to glycans,
also lost the ability to bind to GPIba, is consistent with glycans
contributing the main component of SSL5 binding to GPIba. Whilst
antibody steric hindrance  or the preferential interaction of SSL5
with carbohydrate moieties within GPIba antibodies cannot be
formally excluded as an explanation of our observations using
antibodies with different binding epitopes on GPIba, other potential
binding determinants within GPIba may be involved; for example it is
conceivable that the mucin-rich region within the macroglycopeptide
region of GPIba may modulate the binding of SSL5. Further work is
required to examine this point.
site for SSL5 provides, along with GPIba, a mechanistic basis for
understanding SSL5 induced platelet activation, as GPVI is known to
couple with the robust collagen-induced signalling pathway . The
inhibition of SSL5-induced platelet aggregation by the syk kinase
inhibitor, Bay61-3606 and the src inhibitor PP2, supports this
hypothesis. Furthermore, it has previously been shown that these two
glycoproteins and their respective signalling pathways can directly
interact. A functional complex between GPIba and GPVI on platelets
has been reported, and an important role for the sulphated-tyrosine
region GPIba was identified . It is possible that both platelet
receptors may contribute SSL5 binding determinants, consistent with
surface plasmon resonance data and inhibition of SSL5 binding and
activation of platelets by soluble recombinant GPVI.
As both of the glycoproteins identified are characterised byN-and
O-linked carbohydrate-rich extracellular regions [28,29], we sought
glycanarrayundertaken atCoreHofthe ConsortiumforFunctional
Glycomics revealed a specificity of SSL5 for glycans containing the
sLacNac terminus. Interestingly, a similar specificity has been
previously reported for SSL11 . Found on GPIba , the
sLacNac trisaccharide was present in nineteen of the twenty SSL5-
binding glycans that demonstrated the highest affinity in binding.
The binding of SSL5 to platelet GPIba and GPVI, subsequent
induction of platelet activation, and inhibition of specific adhesion
abilities, may represent a pathogenic mechanism of S. aureus
infection. Whilst, further work is required to determine the effects
of this mediator in vivo, the effects in experiments performed in vitro
suggest SSL5 is a unique potential therapeutic target. To this end,
we investigated the inhibitory potential of the identified glycan
residues. Of the glycans tested for their ability to prevent SSL5-
Figure 5. Platelet GPVI receptor was identified as a novel
binding partner for SSL5. (A) Flow cytometry was used to assess the
inhibition of P-selectin expression on human platelets following pre-
incubation of SSL5 with recombinant GPVI. Bar graphs represent the
geometric mean fluorescence intensity (means 6 SEM) across six
individuals (n=6). ***p,0.001 vs SSL5 only. (B) Direct interaction was
confirmed by surface plasmon resonance using GPVI-Fc chimera.
Figure 4. Sulfated-tyrosine residues of GPIba constitute an important binding region for SSL5. (A) Purified SSL5 proteins incubated with
human washed platelet lysates were co-immuno-precipitated by an anti-GPIba mouse mAb (WM23) and subjected to immunoblotting probed with
an HRP-conjugated anti-His antibody (top panel). The filter was re-probed with mouse anti-human GPIba mAb to show GPIba was present in the
immunoprecipitate (bottom panel). Negative controls were mouse IgG1 or without platelet lysates. Directly mixed platelet lysates with SSL5 used as
positive control. (B) Recombinant, affinity-purified Fc fusion proteins of GPIba were immobilized by injection over CM5-streptavidin biosensor chips
and the binding response of SSL5 with GPIba was determined. (C) Representative histogram of SSL5-platelet binding at 3 mg/ml in presence of
vehicle or anti-GPIba antibodies. Washed platelets incubated with vehicle alone or 3 mg/ml of GPIba antibodies (D) AK2 and (E) WM23 and (F) SZ2,
individually, or (G) in combination for 15 minutes at 37uC prior to addition of 0.3–30 mg/ml SSL5 and binding determined by flow cytometry. SZ2, but
not AK2 or WM23, significantly reduced binding, whilst use of all three antibodies in combination further reduced the amount of SSL5 binding. Data
are presented as mean 6 SEM (** P,0.01, *** P,0.001 by paired two-way ANOVA).
SSL5 Binding Platelets
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SSL5 Binding Platelets
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platelet interactions, sLeXwas the most potent, followed by
sLacNac. Sialic acid glycoside demonstrated a non-significant
inhibition of SSL5. This was a surprising result as 16 of the 22 H-
bonds possible between sLeXand SSL5 are present on the much
smaller sialic acid residue , highlighting the important role of
the remaining six residues in the binding of sLeXto SSL5.
Glycan-based therapeutic strategies are advancing towards
clinical applications with sLeXmimetics representing a promising
new class of anti-inflammatory drugs [31,32,33,34]. One such
example, Bimosiamose, is currently in phase II trials in several
disease applications [35,36,37]. We therefore chose to also test
Bimosiamose in this scenario and found its inhibitory efficacy
range to be 100 mM-1 mM, which is equivalent to the currently
used human dose of 5–60 mg/kg.
In summary, we report the association of SSL5 with two platelet
surface receptors, GPIba and GPVI, which results in platelet
activation and aggregation. We could identify several glycan
structures as potential mediators of binding. Through this better
understanding of the mechanisms involved in SSL5-platelet
interactions, we describe novel inhibitory approaches, which are
based on glycan structures. These data warrant further examina-
tion of SSL5 effects on platelets in vivo.
Materials and Methods
A detailed description of the methods is provided in the
expanded Methods section, available in the Supplemental
Materials and Methods S1.
Production of SSL5 and T175P (mutant SSL5)
The cDNA encoding SSL5 was isolated from S. aureus strain
NCTC8325 and cloned into the pHOG21 vector for expression as
a His-tagged fusion protein in E. coli BL21, then purified by Ni-
affinity as described in Supplemental Materials and Methods S1.
Glycan binding specificity by glycomics array
Based on previous reports on SSL proteins [14,15,22], glycans
were mediate binding of SSL5 to glycoproteins. The printed
mammalian glycan microarray at Core H of the Consortium for
Functional Glycomics (Emory University School of Medicine,
Atlanta) was used for screening of glycan-binding protein specificity,
and providing a high-throughput screen for glycans that can bind to
SSL5. Binding of SSL5 to 377 different glycans was evaluated, and
37 glycans were found to bind SSL5 according to stringent criteria
detailed in Supplemental Materials and Methods S1.
Table 1. SSL5 binding glycans as determined by a glycomic array containing 377 different glycans.
Entry Glycan NameMean RFU
17 Neu5Aca2-3Galb1-4GlcNAcb1-2Mana1-3(Neu5Aca2-6Galb1-4GlcNAcb1-2Mana1-6)Manb1-4GlcNAcb1-4GlcNAcb-Sp12 8043
1Glycan contains sLextetrasaccharide (which includes sLacNac trisaccharide).
A ‘hit’ was defined as any glycan wherein the binding signal from fluorescent-labelled SSL5 (200 mg/ml) exceeded two standard deviations more than 10% of the
strongest binding glycan. The best 20 of the 377 hits are shown. Sp0 (ethoxyamine), Sp8 (propyloxyamine), Sp12 (aspartamide) and Sp14 (threonine) represent
glycosidic linkers used to attach the glycan to the microarray surface. RFU = Relative Fluorescence Units.
Figure 6. Inhibition of SSL5-induced platelet activation by sLeX, sLacNac, sialic acid glycoside and bimosiamose. (A) Binding of SSL5
to untreated or neuraminidase-treated platelets; Filled histogram: anti-His-tag antibody alone. (B) Binding of SSL5 in presence of vehicle or 100 mM
sLeX, sLacNac, sialic acid glycoside. (C) Flow cytometry to assess the ability of a range of glycans to inhibit SSL5-induced expression of P-selectin. (D,
E) Bimosiamose inhibited SSL5-induced platelet activation, both anti-P-selectin and PAC-1 binding. Bar graph data are presented as mean 6 SEM
(n=4-6). *p,0.05, *** p,0.01 vs SSL5 or vehicle.
SSL5 Binding Platelets
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Statistical analysis Download full-text
statistical comparisons for the data were performed using ANOVA
(following a Newman–Keuls test) in GraphPad Prism 5.0 Software,
and differences were considered to be significant at p,0.05.
Materials and Methods S1
We would like to thank Core H of the Consortium for Functional
Glycomics (Emory University School of Medicine, Atlanta) for undertaking
the glycan array study to identify the SSL5-binding glycans.
Conceived and designed the experiments: HH PCJA EK EG KP.
Performed the experiments: HH PCJA EK YCC AS ML JS NB DH IA.
Analyzed the data: HH PCJA EK CEH EG KP. Contributed reagents/
materials/analysis tools: GK EG KP. Wrote the paper: HH PCJA EG KP.
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SSL5 Binding Platelets
PLoS ONE | www.plosone.org9April 2011 | Volume 6 | Issue 4 | e19190