Subscribe to RSS
Agonists of toll-like receptor (TLR)2 and TLR4 are unable to modulate platelet activation by adenosine diphosphate and platelet activating factorFinancial support:This work was supported by a British Heart Foundation PhD Studentship, and an MRC Senior Clinical Fellowship (G116/170) to I.S.
Received06 January 2005
Accepted after resubmission26 July 2005
07 December 2017 (online)
Inappropriate platelet activation is a feature of acute and chronic diseases such as disseminated intravascular coagulation (DIC) and atherosclerosis. Since proinflammatory microbial-derived agonists can be involved in the pathogenesis of these diseases, we examined the potential role ofTLR4 (mediating responses to LPS) andTLR2 (which responds to bacterial lipopeptides) in platelet activation. Our data suggested low-level expression of TLR2 andTLR4 on platelets, determined by flow cytometry, and we also observed expression of TLR4 on a megakaryocytic cell line by both flow cytometry and immunohistochemistry. Stimulation of the platelets with the TLR4 agonist LPS, and the synthetic TLR2 agonist Pam3CSK4, resulted in no platelet aggregation, no increase in CD62P surface expression and no increase in the cytosolic concentration of Ca2+. The TLR agonists were also unable to directly activate platelets primed with epinephrine, or pretreated with a low concentration ofADP or PAF. Pretreatment of platelets with LPS or Pam3CSK4 also failed to modulate the platelet response to submaximal concentrations of the classical platelet agonists ADP and PAF. We conclude that theTLR agonists LPS and Pam3CSK4 have no direct effect on platelet activation and that platelet TLRs may be a remnant from megakaryocytes. TLR2 and TLR4 agonists are thought to have a significant role in diseases such as atherosclerosis and DIC, but our research suggests that this is through a mechanism other than direct platelet activation or by modification of platelet responses to other agonists.
- 1 Ross R. Atherosclerosis is an inflammatory disease. Am Heart J 1999; 138: S419-20.
- 2 Frenette PS. et al. Platelets roll on stimulated endotheliumin vivo: an interaction mediated by endothelial P-selectin. Proc Natl Acad Sci USA 1995; 92: 7450-4.
- 3 Massberg S. et al. A critical role of platelet adhesion in the initiation of atherosclerotic lesion formation. J Exp Med 2002; 196: 887-96.
- 4 Wing DA. et al. Model for disseminated intravascular coagulation: bacterial sepsis in rhesus monkeys. J Lab Clin Med 1978; 92: 239-51.
- 5 Hirschfeld M. et al. Cutting edge: repurification of lipopolysaccharide eliminates signaling through both human and murine toll-like receptor 2. J Immunol 2000; 165: 618-22.
- 6 Poltorak A. et al. Genetic and physical mapping of the Lps locus: identification of the toll-4 receptor as a candidate gene in the critical region. Blood Cells Mol Dis 1998; 24: 340-55.
- 7 Qureshi ST. et al. Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4). J Exp Med 1999; 189: 615-25.
- 8 Wyllie DH. et al. Evidence for an accessory protein function for Toll-like receptor 1 in anti-bacterial responses. J Immunol 2000; 165: 7125-32.
- 9 Ozinsky A. et al. Co-operative induction of pro-inflammatory signaling by Toll-like receptors. J Endotoxin Res 2000; 6: 393-6.
- 10 Takeuchi O. et al. Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins. J Immunol 2002; 169: 10-4.
- 11 Sabroe I. et al. Toll-like receptors: their role in allergy and non-allergic inflammatory disease. Clin Exp Allergy 2002; 32: 984-9.
- 12 Smeeth L. et al. Risk of myocardial infarction and stroke after acute infection or vaccination. N Engl J Med 2004; 351: 2611-8.
- 13 Edfeldt K. et al. Expression of toll-like receptors in human atherosclerotic lesions: a possible pathway for plaque activation. Circulation 2002; 105: 1158-61.
- 14 Kiechl S. et al. Toll-like receptor 4 polymorphisms and atherogenesis. N Engl J Med 2002; 347: 185-92.
- 15 Vink A. et al. In vivo evidence for a role of toll-like receptor 4 in the development of intimal lesions. Circulation 2002; 106: 1985-90. Ward et al.: TLR agonists fail to modulate platelet activation 838
- 16 Hollestelle SC. et al. Toll-like receptor 4 is involved in outward arterial remodeling. Circulation 2004; 109: 393-8.
- 17 Oyama J. et al. Reduced myocardial ischemia-reperfusion injury in toll-like receptor 4-deficient mice. Circulation 2004; 109: 784-9.
- 18 Ream VI. et al. The aggregation of human platelets by bacterial endotoxin. J Lab Clin Med 1965; 66: 245-52.
- 19 Mueller-Eckhardt C, Luscher EF. Immune reactions of human blood platelets. IV. Investigations on the problem of an immunologically induced effect of endotoxin on human platelets. Thromb Diath Haemorrh 1968; 20: 336-44.
- 20 Matera C. et al. Effects of tetanus toxin, Salmonella typhimurium porin, and bacterial lipopolysaccharide on platelet aggregation. J Med 1992; 23: 327-38.
- 21 Saba HI. et al. Endotoxin-mediated inhibition of human platelet aggregation. Thromb Res 1984; 34: 19-33.
- 22 Sheu JR. et al. Mechanisms involved in the antiplatelet activity of Escherichia coli lipopolysaccharide in human platelets. Br J Haematol 1998; 103: 29-38.
- 23 Montrucchio G. et al. Mechanisms of the priming effect of low doses of lipopoly-saccharides on leukocyte-dependent platelet aggregation in whole blood. Thromb Haemost 2003; 90: 872-81.
- 24 Storey RF. et al. The central role of the P(2T) receptor in amplification of human platelet activation, aggregation, secretion and procoagulant activity. Br J Haematol 2000; 110: 925-34.
- 25 Takeuchi K. al. Production of platelet-like particles by a human megakaryoblastic leukemia cell line (MEG-01). Exp Cell Res 1991; 193: 223-6.
- 26 Clarke MC. et al. Compartmentalized megakaryocyte death generates functional platelets committed to caspase-independent death. J Cell Biol 2003; 160: 577-87.
- 27 Ogura M. et al. Establishment of a novel human megakaryoblastic leukemia cell line, MEG-01, with positive Philadelphia chromosome. Blood 1985; 66: 1384-92.
- 28 Corash L. Measurement of platelet activation by fluorescence-activated flow cytometry. Blood Cells 1990; 16: 97-108 discussion 107–8.
- 29 Aderem AA. et al. Bacterial lipopolysaccharides prime macrophages for enhanced release of arachidonic acid metabolites. J Exp Med 1986; 164: 165-79.
- 30 Surette ME. et al. Priming of human peripheral blood mononuclear cells with lipopolysaccharides for enhanced arachidonic acid release and leukotriene synthesis. J Leukoc Biol 1996; 59: 709-15.
- 31 Guthrie LA. et al. Priming of neutrophils for enhanced release of oxygen metabolites by bacterial lipopolysaccharide. Evidence for increased activity of the superoxide-producing enzyme. J Exp Med 1984; 160: 1656-71.
- 32 Born GV. et al. Potentiation of platelet aggregation by adrenaline. J Physiol 1967; 191: 43P-44P.
- 33 Martin DS, Cassisi NJ, Pickens JL. Endotoxin shock: a collective review. Rev Surg 1965; 22: 311-9.
- 34 De Kimpe SJ. et al. The cell wall components peptidoglycan and lipoteichoic acid from Staphylococcus aureus act in synergy to cause shock and multiple organ failure. Proc Natl Acad Sci U S A 1995; 92: 10359-63.
- 35 Sabroe I. et al. Toll-like receptor (TLR)2 and TLR4 in human peripheral blood granulocytes: a critical role for monocytes in leukocyte lipopolysaccharide responses. J Immunol 2002; 168: 4701-10.
- 36 Cognasse F. et al. Evidence of Toll-like receptor molecules on human platelets. Immunol Cell Biol 2005; 83: 196-8.
- 37 Andonegui G, Kerfoot SM, McNagny K. et al. Platelets express functional toll-like receptor-4 (TLR4). Blood. 2005 10.1182/blood-2005–03–0916.
- 38 Shiraki R. et al. Expression of Toll-like receptors on human platelets. Thromb Res 2004; 113: 379-85.
- 39 Hoshino K. et al. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol 1999; 162: 3749-52.
- 40 Shimazu R. et al. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor. J Exp Med 1999; 189: 1777-82.
- 41 da Silva, Correia J. et al. Lipopolysaccharide is in close proximity to each of the proteins in its membrane receptor complex. transfer from CD14 to TLR4 and MD-2. J Biol Chem 2001; 276: 21129-35.
- 42 Wright SD. et al. Lipopolysaccharide (LPS) binding protein opsonizes LPS-bearing particles for recognition by a novel receptor on macrophages. J Exp Med 1989; 170: 1231-41.