Distinct effects of Re- and S-forms of LPS on modulating platelet activation
2013; Elsevier BV; Volume: 11; Issue: 4 Linguagem: Inglês
10.1111/jth.12151
ISSN1538-7933
AutoresJános Kappelmayer, Ildikó Beke Debreceni, András Vida, Péter Antal‐Szalmás, KJ Clemetson, Béla Nagy,
Tópico(s)Platelet Disorders and Treatments
ResumoSepsis is characterized by a robust cellular response to invading organisms. In the case of Gram-negative bacteria it is mostly governed by lipopolysaccharide (LPS), the main outer surface component, which is a strong stimulator of the immune system 1.Hotchkiss R.S. Karl I.E. The pathophysiology and treatment of sepsis.N Engl J Med. 2003; 348: 138-50Crossref PubMed Scopus (3186) Google Scholar. Patients with sepsis frequently show severe thrombocytopenia because of sequestration of activated platelets in the microvasculature 1.Hotchkiss R.S. Karl I.E. The pathophysiology and treatment of sepsis.N Engl J Med. 2003; 348: 138-50Crossref PubMed Scopus (3186) Google Scholar, 2.Andonegui G. Kerfoot S.M. McNagny K. Ebbert K.V. Patel K.D. Kubes P. Platelets express functional Toll-like receptor-4.Blood. 2005; 106: 2417-23Crossref PubMed Scopus (393) Google Scholar. In vitro studies 2.Andonegui G. Kerfoot S.M. McNagny K. Ebbert K.V. Patel K.D. Kubes P. Platelets express functional Toll-like receptor-4.Blood. 2005; 106: 2417-23Crossref PubMed Scopus (393) Google Scholar, 3.Stahl A.L. Svensson M. Mörgelin M. Svanborg C. Tarr P.I. Mooney J.C. Watkins S.L. Johnson R. Karpman D. Lipopolysaccharide from enterohemorrhagic Escherichia coli binds to platelets through TLR4 and CD62 and is detected on circulating platelets in patients with hemolytic uremic syndrome.Blood. 2006; 108: 167-76Crossref PubMed Scopus (0) Google Scholar, 4.Zhang G. Han J. Welch E.J. Ye R.D. Voyno-Yasenetskaya T.A. Malik A.B. Du X. Li Z. Lipopolysaccharide stimulates platelet secretion and potentiates platelet aggregation via TLR4/MyD88 and the cGMP-dependent protein kinase pathway.J Immunol. 2009; 182: 7997-8004Crossref PubMed Scopus (270) Google Scholar, 5.Hashimoto K. Jayachandran M. Owen W.G. Miller V.M. Aggregation and microparticle production through toll-like receptor 4 activation in platelets from recently menopausal women.J Cardiovasc Pharmacol. 2009; 54: 57-62Crossref PubMed Scopus (19) Google Scholar, 6.Cognasse F. Hamzeh-Cognasse H. Lafarge S. Delezay O. Pozzetto B. McNicol A. Garraud O. Toll-like receptor 4 ligand can differentially modulate the release of cytokines by human platelets.Br J Haematol. 2008; 141: 84-91Crossref PubMed Scopus (110) Google Scholar, 7.Ward J.R. Bingle L. Judge H.M. Brown S.B. Storey R.F. Whyte M.K. Dower S.K. Buttle D.J. Sabroe I. Agonists of toll-like receptor (TLR)2 and TLR4 are unable to modulate platelet activation by adenosine diphosphate and platelet activating factor.Thromb Haemost. 2005; 94: 831-8PubMed Google Scholar, 8.Kalsch T. Elmas E. Nguyen X.D. Suvajac N. Klüter H. Borggrefe M. Dempfle C.E. Endotoxin-induced effects on platelets and monocytes in an in vivo model of inflammation.Basic Res Cardiol. 2007; 102: 460-6Crossref PubMed Scopus (46) Google Scholar extensively examined platelet activation induced by a wide range of concentrations of LPS from Escherichia coli strains under rather variable conditions. Surprisingly, the platelet responses gave controversial data, and it was also debated whether LPS directly modulates platelet activation or only in a leukocyte-dependent manner (reviewed in 9.Cox D. Kerrigan S.W. Watson S.P. Platelets and the innate immune system: mechanisms of bacterial-induced platelet activation.J Thromb Haemost. 2011; 9: 1097-107Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Various toll-like receptors (TLR 1, 2, 4, 6, 8 and 9) were detected on the platelet surface, but TLR4 was the main receptor for direct interactions of LPS with platelets 2.Andonegui G. Kerfoot S.M. McNagny K. Ebbert K.V. Patel K.D. Kubes P. Platelets express functional Toll-like receptor-4.Blood. 2005; 106: 2417-23Crossref PubMed Scopus (393) Google Scholar, 3.Stahl A.L. Svensson M. Mörgelin M. Svanborg C. Tarr P.I. Mooney J.C. Watkins S.L. Johnson R. Karpman D. Lipopolysaccharide from enterohemorrhagic Escherichia coli binds to platelets through TLR4 and CD62 and is detected on circulating platelets in patients with hemolytic uremic syndrome.Blood. 2006; 108: 167-76Crossref PubMed Scopus (0) Google Scholar, 6.Cognasse F. Hamzeh-Cognasse H. Lafarge S. Delezay O. Pozzetto B. McNicol A. Garraud O. Toll-like receptor 4 ligand can differentially modulate the release of cytokines by human platelets.Br J Haematol. 2008; 141: 84-91Crossref PubMed Scopus (110) Google Scholar, 9.Cox D. Kerrigan S.W. Watson S.P. Platelets and the innate immune system: mechanisms of bacterial-induced platelet activation.J Thromb Haemost. 2011; 9: 1097-107Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar. Certain clinically relevant Gram-negative bacteria produce a highly heterogeneous mixture of total LPS with varying numbers of repeating polysaccharides 10.Huber M. Kalis C. Keck S. Jiang Z. Georgel P. Du X. Shamel L. Sovath S. Mudd S. Beutler B. Galanos C. Freudenberg M.A. R-form LPS, the master key to the activation of TLR4/MD-2-positive cells.Eur J Immunol. 2006; 36: 701-11Crossref PubMed Scopus (0) Google Scholar. It contains diverse proportions of S-LPS, which comprises lipid A moiety, core-oligosaccharide, and O-polysaccharides, and the rough form of LPS (Re-LPS) lacking O-specific chains. Smooth LPS (S-LPS) from E. coli showed discrepancies in activating platelets 4.Zhang G. Han J. Welch E.J. Ye R.D. Voyno-Yasenetskaya T.A. Malik A.B. Du X. Li Z. Lipopolysaccharide stimulates platelet secretion and potentiates platelet aggregation via TLR4/MyD88 and the cGMP-dependent protein kinase pathway.J Immunol. 2009; 182: 7997-8004Crossref PubMed Scopus (270) Google Scholar, 5.Hashimoto K. Jayachandran M. Owen W.G. Miller V.M. Aggregation and microparticle production through toll-like receptor 4 activation in platelets from recently menopausal women.J Cardiovasc Pharmacol. 2009; 54: 57-62Crossref PubMed Scopus (19) Google Scholar, 6.Cognasse F. Hamzeh-Cognasse H. Lafarge S. Delezay O. Pozzetto B. McNicol A. Garraud O. Toll-like receptor 4 ligand can differentially modulate the release of cytokines by human platelets.Br J Haematol. 2008; 141: 84-91Crossref PubMed Scopus (110) Google Scholar, 7.Ward J.R. Bingle L. Judge H.M. Brown S.B. Storey R.F. Whyte M.K. Dower S.K. Buttle D.J. Sabroe I. Agonists of toll-like receptor (TLR)2 and TLR4 are unable to modulate platelet activation by adenosine diphosphate and platelet activating factor.Thromb Haemost. 2005; 94: 831-8PubMed Google Scholar, 8.Kalsch T. Elmas E. Nguyen X.D. Suvajac N. Klüter H. Borggrefe M. Dempfle C.E. Endotoxin-induced effects on platelets and monocytes in an in vivo model of inflammation.Basic Res Cardiol. 2007; 102: 460-6Crossref PubMed Scopus (46) Google Scholar, while Re-LPS from Salmonella minnesota (Re595) stimulated TLR4/MD2 expressing mast cells 10.Huber M. Kalis C. Keck S. Jiang Z. Georgel P. Du X. Shamel L. Sovath S. Mudd S. Beutler B. Galanos C. Freudenberg M.A. R-form LPS, the master key to the activation of TLR4/MD-2-positive cells.Eur J Immunol. 2006; 36: 701-11Crossref PubMed Scopus (0) Google Scholar, and human monocytes and neutrophils with a potency similar to its parent S-LPS 11.Gomes N.E. Brunialti M.K. Mendes M.E. Freudenberg M. Galanos C. Salomão R. Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood.Braz J Med Biol Res. 2010; 43: 853-8Crossref PubMed Scopus (55) Google Scholar. Thus, we studied for the first time whether Re-LPS (Re595) from S. minnesota alters the levels of various platelet activation markers in platelet-rich plasma (PRP) samples, and compared these data with experiments with the same type of S-LPS from E. coli (O111:B4) used earlier 4.Zhang G. Han J. Welch E.J. Ye R.D. Voyno-Yasenetskaya T.A. Malik A.B. Du X. Li Z. Lipopolysaccharide stimulates platelet secretion and potentiates platelet aggregation via TLR4/MyD88 and the cGMP-dependent protein kinase pathway.J Immunol. 2009; 182: 7997-8004Crossref PubMed Scopus (270) Google Scholar, 5.Hashimoto K. Jayachandran M. Owen W.G. Miller V.M. Aggregation and microparticle production through toll-like receptor 4 activation in platelets from recently menopausal women.J Cardiovasc Pharmacol. 2009; 54: 57-62Crossref PubMed Scopus (19) Google Scholar, 6.Cognasse F. Hamzeh-Cognasse H. Lafarge S. Delezay O. Pozzetto B. McNicol A. Garraud O. Toll-like receptor 4 ligand can differentially modulate the release of cytokines by human platelets.Br J Haematol. 2008; 141: 84-91Crossref PubMed Scopus (110) Google Scholar, 7.Ward J.R. Bingle L. Judge H.M. Brown S.B. Storey R.F. Whyte M.K. Dower S.K. Buttle D.J. Sabroe I. Agonists of toll-like receptor (TLR)2 and TLR4 are unable to modulate platelet activation by adenosine diphosphate and platelet activating factor.Thromb Haemost. 2005; 94: 831-8PubMed Google Scholar, 8.Kalsch T. Elmas E. Nguyen X.D. Suvajac N. Klüter H. Borggrefe M. Dempfle C.E. Endotoxin-induced effects on platelets and monocytes in an in vivo model of inflammation.Basic Res Cardiol. 2007; 102: 460-6Crossref PubMed Scopus (46) Google Scholar. First, to evaluate the biological activity of these LPS forms, we tested their procoagulant properties in a recalcified, LPS-stimulated mononuclear cell suspension. Re-LPS and S-LPS (10 μg mL−1) significantly (P<0.05) decreased the mean clotting time (56.0 s and 60.8 s, respectively) compared with the non-activated control (111.7 s) (Fig. 1A). LPS interacted directly with platelets as FITC-labeled Re-LPS binding resulted in significantly increased FL-1 mean fluorescence intensity (MFI) of the positive cells (MFI, 49.7 ± 8.3; P=0.01) compared with control platelets with unlabeled Re-LPS (10.2 ± 2.4). An even larger increase in MFI (112.4 ± 31) was observed with TRAP (10 μm), as similarly shown 3.Stahl A.L. Svensson M. Mörgelin M. Svanborg C. Tarr P.I. Mooney J.C. Watkins S.L. Johnson R. Karpman D. Lipopolysaccharide from enterohemorrhagic Escherichia coli binds to platelets through TLR4 and CD62 and is detected on circulating platelets in patients with hemolytic uremic syndrome.Blood. 2006; 108: 167-76Crossref PubMed Scopus (0) Google Scholar where CD62P was also implicated in LPS binding (Fig. 1B). This 'valid' fluorescence was abolished by a 20-fold molar excess of unlabeled Re-LPS (Fig. S1A), and considerably decreased by anti-TLR4 and anti-P-selectin antibodies (Fig. S1B), in agreement with previous results 3.Stahl A.L. Svensson M. Mörgelin M. Svanborg C. Tarr P.I. Mooney J.C. Watkins S.L. Johnson R. Karpman D. Lipopolysaccharide from enterohemorrhagic Escherichia coli binds to platelets through TLR4 and CD62 and is detected on circulating platelets in patients with hemolytic uremic syndrome.Blood. 2006; 108: 167-76Crossref PubMed Scopus (0) Google Scholar. Next, we examined whether Re-LPS and S-LPS affected platelet aggregation (Fig. 1C-D). Neither LPS form alone (0.1–10 μg mL−1) induced platelet aggregation, even with pretreatment with LPS for 10–60 min, or when only added to the PRP sample prior to recording 4.Zhang G. Han J. Welch E.J. Ye R.D. Voyno-Yasenetskaya T.A. Malik A.B. Du X. Li Z. Lipopolysaccharide stimulates platelet secretion and potentiates platelet aggregation via TLR4/MyD88 and the cGMP-dependent protein kinase pathway.J Immunol. 2009; 182: 7997-8004Crossref PubMed Scopus (270) Google Scholar. In contrast, 1 μg mL−1 of Re-LPS but not S-LPS augmented submaximal TRAP-induced (5 μm) platelet aggregation. The expression of CD62P and CD40L as sensitive activation markers 12.Nagy Jr, B. Miszti-Blasius K. Kerényi A. Clemetson K.J. Kappelmayer J. Potential therapeutic targeting of platelet-mediated cellular interactions in atherosclerosis and inflammation.Curr Med Chem. 2012; 19: 518-31Crossref PubMed Scopus (0) Google Scholar was also analyzed after activation with up to 10 μg mL−1 of Re-LPS and S-LPS in parallel. No increase in the level of P-selectin-positive platelets was induced by either LPS form compared with the untreated sample (9.0 ± 4.0%, 9.5 ± 5.0% vs. 7.6 ± 3.0%; data not shown). On the other hand, a statistically significant elevation in CD40L expression (2.7 ± 1.9%; P<0.05) with high MFI values (172.5 ± 80.0; P<0.001) was detected but only at 10 μg mL−1 of Re-LPS vs. the negative control (1.3 ± 0.2%; MFI = 21.0 ± 8.0). In addition, TRAP also caused a substantial increase in CD40L expression (33.6 ± 4.9%; MFI = 33.6 ± 4.7). Interestingly, S-LPS did not raise the level of surface CD40L (1.4 ± 0.43%; MFI = 22.0 ± 3.0), in contrast to earlier reports 6.Cognasse F. Hamzeh-Cognasse H. Lafarge S. Delezay O. Pozzetto B. McNicol A. Garraud O. Toll-like receptor 4 ligand can differentially modulate the release of cytokines by human platelets.Br J Haematol. 2008; 141: 84-91Crossref PubMed Scopus (110) Google Scholar, 8.Kalsch T. Elmas E. Nguyen X.D. Suvajac N. Klüter H. Borggrefe M. Dempfle C.E. Endotoxin-induced effects on platelets and monocytes in an in vivo model of inflammation.Basic Res Cardiol. 2007; 102: 460-6Crossref PubMed Scopus (46) Google Scholar (Fig. 1E). We found a significant increase (P<0.05) in platelet-derived microparticle (PMP) levels induced by Re-LPS (10 μg mL−1) (450 ± 170 PMPs μL−1 plasma); however, S-LPS did not alter the PMP number (273 ± 144 PMPs μL−1 plasma vs. 227 ± 95 PMPs μL−1 plasma in unstimulated sample). These levels were below those we observed in TRAP-treated (5 μm) samples (970 ± 145 PMPs μL−1 plasma) (Fig. 1F). We also wondered if Re-LPS at even lower concentrations could potentiate PMP generation in the presence of TRAP by synergism. PMP levels were significantly enhanced already at 1 μg mL−1 of Re-LPS during co-activation (1384 ± 299 PMPs μL−1 plasma; P<0.05 compared with the sample with TRAP alone). Of note, as concluded before 5.Hashimoto K. Jayachandran M. Owen W.G. Miller V.M. Aggregation and microparticle production through toll-like receptor 4 activation in platelets from recently menopausal women.J Cardiovasc Pharmacol. 2009; 54: 57-62Crossref PubMed Scopus (19) Google Scholar, S-LPS had neither synergistic nor inhibitory effects on the PMP level under the same conditions (983 ± 120 PMPs μL−1 plasma). Finally, S-LPS at a high concentration was unable to prevent the effects of Re-LPS (Fig. S1C). This suggests that these forms of LPS interact differently with platelets. It was previously established that, in contrast to S-LPS that needs CD14 to bind to TLR4 for its functions 13.Jiang Z. Georgel P. Du X. Shamel L. Sovath S. Mudd S. Huber M. Kalis C. Keck S. Galanos C. Freudenberg M. Beutler B. CD14 is required for MyD88-independent LPS signaling.Nat Immunol. 2005; 6: 565-70Crossref PubMed Scopus (522) Google Scholar, Re-LPS interacts with TLR4 without CD14 10.Huber M. Kalis C. Keck S. Jiang Z. Georgel P. Du X. Shamel L. Sovath S. Mudd S. Beutler B. Galanos C. Freudenberg M.A. R-form LPS, the master key to the activation of TLR4/MD-2-positive cells.Eur J Immunol. 2006; 36: 701-11Crossref PubMed Scopus (0) Google Scholar, 13.Jiang Z. Georgel P. Du X. Shamel L. Sovath S. Mudd S. Huber M. Kalis C. Keck S. Galanos C. Freudenberg M. Beutler B. CD14 is required for MyD88-independent LPS signaling.Nat Immunol. 2005; 6: 565-70Crossref PubMed Scopus (522) Google Scholar. As platelets do not express CD14 3.Stahl A.L. Svensson M. Mörgelin M. Svanborg C. Tarr P.I. Mooney J.C. Watkins S.L. Johnson R. Karpman D. Lipopolysaccharide from enterohemorrhagic Escherichia coli binds to platelets through TLR4 and CD62 and is detected on circulating platelets in patients with hemolytic uremic syndrome.Blood. 2006; 108: 167-76Crossref PubMed Scopus (0) Google Scholar, we checked whether soluble CD14 is required for Re-LPS to influence platelet activation. Gel-filtered platelets with undetectable levels of plasma proteins were activated with added fibrinogen (2 g L−1) by Re-LPS (1 μg mL−1) with TRAP (5 μm) in the absence or presence of LPS binding protein (LBP) and soluble CD14 (data not shown). Platelet aggregation was not further enhanced when platelets were also pretreated with LBP and soluble CD14 at Re-LPS plus TRAP stimulation. Thus, Re-LPS did not require soluble CD14 to activate platelets. In summary, although both LPS forms are biologically active, Re-LPS, but not S-LPS, directly modulates platelet activation. The authors state that they have no conflict of interest. This project was supported by a grant from the Hungarian National Science Foundation (T75199) (JK), and by the János Bólyai Research Fellowship of the Hungarian Academy of Sciences (BNJr). Download .jpg (.1 MB) Help with files Figure S1. Analysis of Re-LPS binding to platelets (A). Unlabeled Re-LPS at very high concetrations prevented the binding of Re-LPS-FITC to platelets and abolished the MFI value for Re-LPS-FITC in TRAP-activated platelets. Investigation of the role of TLR4 and CD62 receptors in the interaction of platelets and LPS (B). Re-LPS-FITC MFI values were markedly decreased in the presence of anti-TLR4 and anti-CD62 antibodies in TRAP-stimulated platelets compared with the untreated TRAP-activated sample. Analysis of PMP levels for studying the distinct binding properties of Re-LPS and S-LPS (C). S-LPS did not interfere with Re-LPS effects because Re-LPS elevated PMP levels even after preincubation with an excess of S-LPS.
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