Mechanisms of the priming effect of low doses of lipopolysaccharides on leukocyte-dependent platelet aggregation in whole blood

Summary

Several studies focused on the ability of bacterial lipopolysaccharides (LPS) in triggering platelet and/or leukocyte activation. The aim of this study was to investigate the molecular mechanisms involved in the aggregation of platelets and in their interaction with leukocytes in whole blood after stimulation with low doses of LPS.

LPS did not directly induce platelet aggregation in whole blood, but they primed the aggregation of platelets induced by epinephrine, adenosine diphosphate and arachidonic acid. As shown by cytofluorimetry, platelets neither bind FITC-LPS, nor express the LPS-receptors CD14 and toll-like receptor 4 (TLR4). On the contrary, LPS primed monocytes and to a lesser extent polymorphonuclear neutrophils to adhere to platelets. Both platelet-leukocyte interaction and platelet aggregation in whole blood were inhibited by blockade of CD14 and TLR4. Moreover, the interaction between platelets and leukocytes was inhibited by P-selectin, and by blockade of PAF and reactive oxygen species, suggesting a role of P-selectin and of leukocyte-derived mediators.

In conclusion, these results elucidate the mechanisms leading to platelet activation and interaction with leukocytes triggered by LPS. They suggest that the activation of platelets by LPS is mainly dependent on leukocytes and especially monocytes as a result of CD14 and TLR4 engagement. Moreover, we found that leukocyte-platelet interaction was triggered by the synthesis of PAF and the generation of oxygen radicals that induced upregulation of surface expression of P-selectin.

Keywords

Monocytes - platelet-activating factor - radical oxygen - CD14 - P-selectin - TLR-4

• References

• 1 Vincent J-L, Yagushi A, Pradier O. Platelet function in sepsis. Crit Care Med 2002; 30: S313-S317.
• 2 Tapper H, Herwald H. Modulation of hemo-static mechanisms in bacterial infectious diseases. Blood 2000; 96: 2329-37.
• 3 Bone RC. The pathogenesis of sepsis. Ann Intern Med 1991; 115: 457-69.
• 4 Davis RB, Meeker WR, McQuarrie DG. Immediate effects of intravenous endotoxin on serotonin concentrations and blood products. Circ Res 1960; 8: 234-9.
• 5 Neame PB, Kelton JG, Walker IR. et al Thrombocytopenia in septicemia: the role of disseminated intravascular coagulation. Blood 1980; 56: 88-92.
• 6 Stohlawetz P, Folman CC, von dem Borne AE. et al Effects of endotoxemia on thrombopoie-sis in men. Thromb Haemost 1999; 81: 613-7.
• 7 Shibazaki M, Kawabata Y, Yokochi T. et al Complement-dependent accumulation and degradation of platelets in the lung and liver induced by injection of lipopolysaccharides. Infect Immun 1999; 67: 5186-91.
• 8 Baba K. Effects of E. coli LPS on human platelet aggregation. Jpn J Anesthesiol 1994; 43: 339-46.
• 9 Sheu JR, Hung WC, Kan YC. et al Mechanisms involved in the antiplatelet activity of Escherichia coli lipopolysaccharide in human platelets. Br J Haematol 1998; 103: 29-38.
• 10 Wright SD, Ramos RA, Tobias PS. et al CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 1990; 249: 1431-3.
• 11 Aderem A, Ulevitch RJ. Toll-like receptors in the induction of the innate immune response. Nature 2000; 406: 782-7.
• 12 Tapping I R, Akashi S, Miyake K. et al Toll-like receptor 4, but not toll-like receptor 2, is a signaling receptor for Escherichia and Salmonella lipopolysaccharides. J Immunol 2000; 165: 5780-7.
• 13 Csako G, Suba EA, Elin RJ. Endotoxin-induced platelet activation in human whole blood in vitro. Thromb Haemost 1988; 59: 378-82.
• 14 Whitworth NH, Barradas MA, Mikhailidis DP. et al An investigation into the effects of bacterial lipopolysaccharide on human platelets. Eur J Haematol 1989; 43: 112-9.
• 15 Nystrom ML, Barradas MA, Jeremy JY. et al Platelet shape change in whole blood: Differential effects of endotoxin. Thromb Haemost 1994; 71: 646-50.
• 16 Schwartz BS, Monroe MC. Human platelet aggregation is initiated by peripheral blood mononuclear cells exposed to bacterial lipopolysaccharide in vitro. J Clin Invest 1986; 78: 1136-41.
• 17 Altieri DC, Mannucci PM. Thromboxane generation by human monocytes enhances platelet function. J Exp Med 1986; 164: 1815-20.
• 18 Heuer HO, Casals-Stenzel J, Muacevic G. et al Pharmacologic activity of bepafant (WEB 2170), a new and selective hetrazepinoic antagonist of platelet-activating factor. J Pharmacol Exp Ther 1990; 255: 962-70.
• 19 Johnston I G, Cook RG, McEver RP. Cloning of GMP-140, a granule membrane protein of platelets and endothelium: sequence similarity to proteins involved in cell adhesion and inflammation. Cell 1989; 56: 1033-44.
• 20 Shimazu R, Akashi S, Ogata H. et al MD-2, a molecule that confers lipopolysaccharide responsiveness on toll-like receptor 4. J Exp Med 1999; 189: 1777-82.
• 21 Born GVR. Aggregation of blood platelets by adenosine and its reversal. Nature 1962; 194: 927
• 22 Valone FH, Epstein LB. Biphasic platelet-activating factor synthesis by human mono-cytes stimulated with IL-1ß, tumor necrosis factor or IFN-g. J Immunol 1988; 141: 3945-50.
• 23 Camussi G, Tetta C, Bussolino F. et al Synthesis and release of platelet-activating factor is inhibited by plasma alpha1-proteinase inhibitor or by alpha1-antichimotrypsin and is stimulated by proteinases. J Exp Med 1988; 168: 1293-1306.
• 24 Gay JC. Mechanism and regulation of neutrophil priming by platelet-activating factor. J Cell Physiol 1993; 156: 189-97.
• 25 Montrucchio G, Bergerone S, Bussolino F. et al Streptokinase induces intravascular release of platelet-activating factor in patients with acute myocardial infarction and stimulates its synthesis by cultured human endothelial cells. Circulation 1993; 88: 1476-83.
• 26 Heinzelmann M, Mercer-Jones MA, Flodgaard H. et al Heparin-binding protein (CAP37) is internalized in monocytes and increases LPS-induced monocyte activation. J Immunol 1998; 160: 5530-6.
• 27 Rinder HM, Bonan JL, Rinder CS. et al Activated and unactivated platelet adhesion to monocytes and neutrophils. Blood 1991; 78: 1760-9.
• 28 van Deventer SJH, Buller HR, ten Cate JW. et al Endotoxaemia, an early predictor of septicaemia in febrile patients. Lancet 1988; 1 (8656) 605-609.
• 29 Henson P. Interactions between neutrophils and platelets. Lab Invest 1990; 62: 391-3.
• 30 Sabroe I, Jones EC, Uscher LR. 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.
• 31 Aida Y, Pabst MJ. Priming of neutrophils by lipopolysaccharide for enhanced release of superoxide. J Immunol 1990; 145: 3017-25.
• 32 Surette ME, Palmantier R, Gosselin J. et al Lipopolysaccharide prime whole blood and isolated neutrophils for the increased synthesis of 5-lipoxygenase products by enhancing arachidonic acid availability: involvement of the CD14 antigen. J Exp Med 1993; 178: 1347-55.
• 33 Ostrovsky L, King AJ, Bond S. et al A justacrine mechanism for neutrophils adhesion on platelets involves platelet-activating factor and a selectin-dependent activation process. Blood 1998; 91: 3028-36.
• 34 Salvemini D, Botting R. Modulation of platelet function by free radicals and free-radical scavengers. Trends Pharmacol Sci 1993; 14: 36-42.
• 35 Weyrich AS, McIntyre TM, McEver RP. et al Monocyte tethering by P-selectin regulates monocyte chemotactic protein-1 and tumor necrosis factor-alpha secretion. Signal integration and NF-kappa B translocation. J Clin Invest 1995; 95: 2297-2303.
• 36 Nagata K, Tsuji T, Todoroki N. et al Activated platelets induce superoxide anion release by monocytes and neutrophils through P-selectin (CD62). J Immunol 1993; 151: 3267-73.
• 37 Elstad MR, La Pine TR, Cowley FS. et al P-selectin regulates Platelet-Activating Factor synthesis and phagocytosis by monocytes. J Immunol 1995; 155: 2109-22.
• 38 Cerletti C, Evangelista V, de Gaetano G. P-selectin-beta-2-integrin cross-talk: a molecular mechanism for polymorphonuclear leukocyte recruitment at the site of vascular damage. Thromb Haemost 1999; 82: 787-93.
• 39 Weirich AS, Elstad MR, McEver RP. et al Activated platelets signal chemokine synthesis by human monocytes. J Clin Invest 1996; 97: 1525-34.