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Abstract

  1. Top of page
  2. Abstract
  3. Acknowledgement
  4. Disclosure of Conflict of Interests
  5. References
  6. Supporting Information

Armstrong PCJ, Hu H, Rivera J, Rigby S, Chen Y-C, Howden BP, Gardiner E, Peter K. Staphylococcal superantigen-like protein 5 induces thrombotic and bleeding complications in vivo: inhibition by an anti-SSL5 antibody and the glycan Bimosiamose. J Thromb Haemost 2012; 10: 2607–9.

Systemic Staphylococcus (S.) aureus infections are associated with disseminated intravascular coagulation (DIC), a disorder characterized by simultaneous thrombosis and bleeding, which ultimately leads to multiple organ failure and thrombocytopenia [1]. S. aureus secretes pathogenic proteins, including the Staphylococcal superantigen-like proteins (SSLs), a protein family structurally homologous to the superantigens (SAg). Generally considered immunomodulatory, SSLs prevent the adhesion and migration of neutrophils to sites of infection [2] and leukocyte activation by chemokines and anaphylatoxins [3].

We and others have shown that one member, SSL5, directly binds to and activates platelets via interaction with glycoproteins (GP)Ibα and GPVI [4,5]. Here, we investigated the pathophysiological relevance of SSL5 as an important mediator, and thus potential therapeutic target, of S. aureus-induced thromboembolic complications in vivo.

To examine whether SSL5 induces acute thromboembolism, C57BL/6 mice were intravenously administered SSL5 (10 μg g−1 body weight (BW)) for 15 min prior to analysis. Immunohistochemical analyses of lungs (Fig. 1A and Fig. S3) found evidence of platelet-rich (CD41+) thrombi (10 ± 1.8 thrombi), which were absent in vehicle-treated mice (Fig. 1B). This finding reflects the major symptom of DIC [6] and provided us with the rationale to pharmacologically target SSL5 interaction with platelets in vivo.

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Figure 1.  (A) Thrombus formation in the lungs of C57BL/6 mice induced by SSL5 (4 μg g−1 BW), as shown in representative hematoxylin and eosin stained sections from mice administered vehicle (top left), SSL5 (top right) and SSL5 co-administered with 5E3 (8 μg g−1 BW; bottom left) or Bimosiamose (3.5 μg g−1 BW; bottom right), magnification × 100. (B) Quantification of SSL5-induced lung thrombi and inhibition by Bimosiamose (3.5 μg g−1BW) and 5E3 (8 μg g−1 BW), n = 4–5 per group. (C) Reduction of platelet adhesion by Bimosiamose (500 μm) in flow chamber experiments with reconstituted blood, flowing (300 s−1) through a SSL5-coated (1 μm) capillary for 5 min, n = 5 independent experiments. (D) Inhibition of SSL5-induced platelet activation by 5E3 in a concentration-dependent manner as measured by P-selectin expression, n = 3 independent experiments. (E) Effect of SSL5 (10 μg mL−1) on platelet adhesion following static incubation in VWF-coated wells with or without pre-activation with ADP (20 μm), n = 7 independent experiments. (F) SSL5 (10 μg g−1 BW) prolonged mouse tail bleeding time compared with vehicle; this was completely ablated when co-administered with Bimosiamose (3.5 μg g−1 BW) or 5E3 (8 μg g−1 BW), n = 7–15 per group. Data presented as mean ± SEM, *< 0.05; **< 0.01; ***< 0.001, by Student’s t-test or one-way anova with Bonferroni post-test as appropriate.

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Our identification of key glycan motifs mediating SSL5-receptor interactions led us to test a glycan-based therapeutic, Bimosiamose (Revotar, Hennigsdorf, Germany), which we hypothesized would compete with glycoproteins for SSL5 binding. We have demonstrated that Bimosiamose prevents SSL5-induced platelet activation during static incubation [4]. DeHaas et al. [5] have shown adhesion of platelets to SSL5 under flow in reconstituted blood and we used this model to show that Bimosiamose (500 μm) inhibits platelets adhering to SSL5 (Fig. 1C) whilst under flow (5 min, 300 s−1).

As an alternative therapeutic approach, we developed a monoclonal anti-SSL5 antibody (5E3), which displays strong SSL5 specificity over SSL7 and SSL11 (data not shown), SSL proteins with the highest structural homogeneity to SSL5 [7]. 5E3 blocked SSL5-induced P-selectin expression, a marker of platelet activation, in a concentration-dependent manner (Fig. 1D), but did not inhibit ADP-induced platelet activation (Fig. S1). Flow cytometric analysis confirmed that Bimosiamose and 5E3 directly prevented SSL5 binding to platelets (Fig. S2).

We next tested Bimosiamose and 5E3 as therapeutic approaches in vivo. Compared with SSL5 alone (4 μg g−1 BW; 10 ± 1.8), co-administration with Bimosiamose (3.5 μg g−1 BW; 1.5 ± 0.3; < 0.001) or 5E3 (8 μg g−1 BW; 2.9 ± 0.6; < 0.01) significantly reduced the number of thrombi (Fig. 1B).

Bleeding complications are a major characteristic feature of DIC. Von Willebrand factor (VWF) is a key ligand mediating platelet rolling and contact adhesion via the platelet glycoprotein GPIbα, part of the GPIb-IX-V complex [8]. This interaction is especially important in the initial stages of the thrombotic response and disruption may cause increased bleeding. SSL5 binds to the sulfated tyrosine region of GPIbα [4], a key VWF-binding motif, and thus in addition to activating platelets, may inhibit GPIbα-vWF binding.

SSL5 activates platelets and therefore on its own, can increase adhesion of platelets to VWF under static conditions (Fig. 1E). However, at the same time, SSL5 can have an inhibitory effect on the adhesion of ADP-activated platelets (Fig. 1E; ∼48%; < 0.05). This indicates that when platelets are already activated, SSL5 has an independent inhibiting effect on platelet adhesion. We used a mouse tail-cut bleeding assay to test the potential functional consequence of this inhibition in vivo. SSL5 (10 μg g−1 BW) administered 10 min prior to analysis increased bleeding time (588 ± 72 s) compared with vehicle (147 ± 23 s; < 0.05; Fig. 1F). Both Bimosiamose (224 ± 64 s) and 5E3 (269 ± 39 s) significantly reduced bleeding times compared with SSL5 alone (Fig. 1F).

This study is the first to implicate SSL5 as a mediator of thrombotic and bleeding complications in vivo. Indeed, anti-SSL5 antibodies have been detected in human sera [9], highlighting exposure to SSL5 during S. aureus infection in humans. Moreover, our findings show that two structurally different and novel inhibitory approaches, one glycan based the other antibody based, reverse the effects of SSL5 in vivo. Future investigations using an animal model of S. aureus infection are warranted to validate whether these two compounds have an inhibitory effect in a clinically relevant setting, and may provide insight into additional mechanisms by which SSL5 may contribute to sepsis-induced DIC (e.g. platelet-neutrophil aggregate formation and organ dysfunction).

Mortality rates in S. aureus sepsis are around 20%, and increase above 40% upon developing DIC [10]. Therefore, there is an enormous clinical need to prevent S. aureus-induced DIC by identifying novel therapeutic strategies. Antibody-based therapeutics currently represents the majority of new drug development programs [11], and thus 5E3 is a promising template for further drug development. The concentrations of Bimosiamose used in our study compare well with doses well tolerated in animal studies [12]. Bimosiamose is currently undergoing phase II clinical trials as an anti-inflammatory drug [13], indicating that this glycan-based therapeutic is highly translational and may be rapidly available for clinical use.

In summary, our studies indicate that SSL5 may play a causative role in thrombotic and bleeding complications associated with S. aureus infections and represents a potential therapeutic target. We also demonstrate the therapeutic potential of Bimosiamose and our 5E3 monoclonal antibody, which warrants further examination.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Acknowledgement
  4. Disclosure of Conflict of Interests
  5. References
  6. Supporting Information

We gratefully acknowledge the contribution of G. Krippner in obtaining Bimosiamose.

Disclosure of Conflict of Interests

  1. Top of page
  2. Abstract
  3. Acknowledgement
  4. Disclosure of Conflict of Interests
  5. References
  6. Supporting Information

The authors state that they have no conflict of interests.

References

  1. Top of page
  2. Abstract
  3. Acknowledgement
  4. Disclosure of Conflict of Interests
  5. References
  6. Supporting Information
  • 1
    Takemitsu T, Wada H, Hatada T, Ohmori Y, Ishikura K, Takeda T, Sugiyama T, Yamada N, Maruyama K, Katayama N, Isaji S, Shimpo H, Kusunoki M, Nobori T. Prospective evaluation of three different diagnostic criteria for disseminated intravascular coagulation. Thromb Haemost 2011; 105: 404.
  • 2
    Bestebroer J, Poppelier MJ, Ulfman LH, Lenting PJ, Denis CV, van Kessel KP, van Strijp JA, de Haas CJ. Staphylococcal superantigen-like 5 binds PSGL-1 and inhibits P-selectin-mediated neutrophil rolling. Blood 2007; 109: 293643.
  • 3
    Bestebroer J, van Kessel KP, Azouagh H, Walenkamp AM, Boer IG, Romijn RA, van Strijp JA, de Haas CJ. Staphylococcal SSL5 inhibits leukocyte activation by chemokines and anaphylatoxins. Blood 2009; 113: 32837.
  • 4
    Hu H, Armstrong PC, Khalil E, Chen YC, Straub A, Li M, Soosairajah J, Hagemeyer CE, Bassler N, Huang D, Ahrens I, Krippner G, Gardiner E, Peter K. GPVI and GPIbalpha mediate staphylococcal superantigen-like protein 5 (SSL5) induced platelet activation and direct toward glycans as potential inhibitors. PLoS ONE 2011; 6: e19190.
  • 5
    de Haas CJ, Weeterings C, Vughs MM, de Groot PG, van Strijp JA, Lisman T. Staphylococcal superantigen-like 5 activates platelets and supports platelet adhesion under flow conditions, which involves glycoprotein Ibalpha and alpha IIb beta 3. J Thromb Haemost 2009; 7: 186774.
  • 6
    Levi M, Toh CH, Thachil J, Watson HG. Guidelines for the diagnosis and management of disseminated intravascular coagulation. British Committee for Standards in Haematology. Br J Haematol 2009; 145: 2433.
  • 7
    Chung MC, Wines BD, Baker H, Langley RJ, Baker EN, Fraser JD. The crystal structure of staphylococcal superantigen-like protein 11 in complex with sialyl Lewis X reveals the mechanism for cell binding and immune inhibition. Mol Microbiol 2007; 66: 134255.
  • 8
    Andrews RK, Gardiner EE, Shen Y, Whisstock JC, Berndt MC. Glycoprotein Ib-IX-V. Int J Biochem Cell Biol 2003; 35: 11704.
  • 9
    Arcus VL, Langley R, Proft T, Fraser JD, Baker EN. The three-dimensional structure of a superantigen-like protein, SET3, from a pathogenicity island of the Staphylococcus aureus genome. J Biol Chem 2002; 277: 3227481.
  • 10
    Martin GS, Mannino DM, Eaton S, Moss M. The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med 2003; 348: 154654.
  • 11
    Hagemeyer CE, Peter K. Targeting the platelet integrin GPIIb/IIIa. Curr Pharm Des 2010; 16: 411933.
  • 12
    Hicks AE, Abbitt KB, Dodd P, Ridger VC, Hellewell PG, Norman KE. The anti-inflammatory effects of a selectin ligand mimetic, TBC-1269, are not a result of competitive inhibition of leukocyte rolling in vivo. J Leukoc Biol 2005; 77: 5966.
  • 13
    Beeh KM, Beier J, Meyer M, Buhl R, Zahlten R, Wolff G. Bimosiamose, an inhaled small-molecule pan-selectin antagonist, attenuates late asthmatic reactions following allergen challenge in mild asthmatics: a randomized, double-blind, placebo-controlled clinical cross-over-trial. Pulm Pharmacol Ther 2006; 19: 23341.

Supporting Information

  1. Top of page
  2. Abstract
  3. Acknowledgement
  4. Disclosure of Conflict of Interests
  5. References
  6. Supporting Information

Fig. S1. No inhibition by 5E3 mAb (5–50 μg mL−1) of ADP (20 μM)- induced P-selectin expression, a marker of platelet activation, as measured by flow cytometry.

Fig. S2. 5E3 mAb (50 μg mL−1) and Bimosiamose (1 mM) directly prevent SSL5 (10 μg mL−1)-binding to platelets.

Fig. S3. Representative lung sections from mice administered SSL5 (4 μg g−1; i.v.) intravenously.

Data S1. Methods and Materials.

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JTH_12022_sm_FigS1-S3.pdf177KSupporting info item
JTH_12022_sm_Methods-and-Materials.doc54KSupporting info item

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