GPVI and GPIbα mediate staphylococcal superantigen-like protein 5 (SSL5) induced platelet activation and direct toward glycans as potential inhibitors

Abstract

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 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.

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.

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 µg/ml SSL5 or SSL5 mutant T175P. (C) Washed platelets were incubated with 0.1–80 µg/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 ± SEM of three independent experiments.

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 µM ADP, 100 ng/ml convulxin, or 30 µg/ml SSL5 or T175P SSL5. (C) Representative aggregation trace obtained in washed platelets, induced by 20 µg/ml SSL5 or no stimulation (PBS). Aggregation was strongly inhibited by pre-treatment of platelet with either a syk kinase inhibitor (BAY61-3606, 5 µM), or a src inhibitor (PP2, 3 µM). Images are representative of three independent experiments. * P<0.05.

Figure 3. SSL5 induces spreading of platelets on fibrinogen.

Gel filtered platelets (GFP) were incubated with 20 µM ADP, 1–10 µg/ml SSL5, 10 µg/ml T175P (mutant SSL5) at 37°C for 30 minutes on fibrinogen. (A) Representative pictures were obtained by DIC (×60) following adhesion. (B) Presented data are means ± SEM of four independent experiments using five separate fields per experiment. ** P<0.01, *** P<0.001 compared to control (PBS).

Figure 4. Sulfated-tyrosine residues of GPIbα constitute an important binding region for SSL5.

(A) Purified SSL5 proteins incubated with human washed platelet lysates were co-immuno-precipitated by an anti-GPIbα 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 GPIbα mAb to show GPIbα 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 GPIbα were immobilized by injection over CM5-streptavidin biosensor chips and the binding response of SSL5 with GPIbα was determined. (C) Representative histogram of SSL5-platelet binding at 3 µg/ml in presence of vehicle or anti-GPIbα antibodies. Washed platelets incubated with vehicle alone or 3 µg/ml of GPIbα antibodies (D) AK2 and (E) WM23 and (F) SZ2, individually, or (G) in combination for 15 minutes at 37°C prior to addition of 0.3–30 µg/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 ± SEM (** P<0.01, *** P<0.001 by paired two-way ANOVA).

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 ± 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 6. Inhibition of SSL5-induced platelet activation by sLe^X, 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 µM sLe^X, 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 ± SEM (n = 4-6). *p<0.05, *** p<0.01 vs SSL5 or vehicle.