Thrombelastography for the monitoring of lipopolysaccharide induced activation of coagulation

Kai Zacharowski; Christoph Sucker; Paula Zacharowski; Matthias Hartmann

doi:10.1160/TH05-06-0420

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2006; 95(03): 557-561
DOI: 10.1160/TH05-06-0420

New Technologies, Diagnostic Tools and Drugs

Schattauer GmbH

Thrombelastography for the monitoring of lipopolysaccharide induced activation of coagulation

Kai Zacharowski^*

¹Department of Anesthesiology, University Hospital Düsseldorf, Germany

²Molecular Cardioprotection and Inflammation Group, University Hospital Düsseldorf, Germany

,

Christoph Sucker^*

³Department of Haemostasis and Transfusion Medicine, University Hospital Düsseldorf, Germany

,

Paula Zacharowski

¹Department of Anesthesiology, University Hospital Düsseldorf, Germany

²Molecular Cardioprotection and Inflammation Group, University Hospital Düsseldorf, Germany

,

Matthias Hartmann

¹Department of Anesthesiology, University Hospital Düsseldorf, Germany

› Author Affiliations

Abstract

Summary

During Gram-negative sepsis, lipopolysaccharide (LPS) activates toll-like receptor (TLR) 4 and induces complex responses of immune system and haemostasis. In the present study we investigated whether thrombelastography is suitable to monitor the LPS-induced activation of coagulation. Whole blood samples from healthy volunteers were incubated with LPS for various in-cubation periods (0–5 hrs), thereafter rotation thrombelastography was performed. Incubation of whole blood (≥ 3 h) with LPS markedly reduced clotting time; after 5 hrs the variable was reduced from 459 ± 39 sec to 80 ± 20 sec while the other thrombelastography variables (angle α, clot formation time, maximal clot formation) remained unaltered. EC⁵⁰ of the LPSeffect on whole blood clotting time was 18 µg/ml.In isolated leu kocytes, diluted in platelet poor plasma, far lower LPS-concentrations were effective: 10 ng/ml LPS reduced clotting time from 439 ± 68 sec to 200± 56 sec. Experiments with the protein synthesis inhibitor cycloheximide and active site-inhibited factor VIIa revealed that LPS exerts its effects via the synthesis of tissue factor.Addition of tissue factor to whole blood samples revealed that a concentration of 100 fmol/l can be detected using thrombelastography. In whole blood samples the tissue factor concentration induced by LPS amounted up to 12 pmol/l. In summary, thrombelastography proved to bea sensitive and reliable tool for the determination of LPS-induced tissue factor mediated activation of haemostasis in whole blood samples.

Keywords

Human whole blood - lipopolysaccharide - thrombelastography

Full Text

References

References
1 Opal SM. Interactions between coagulation and inflammation. Scand J Infect Dis 2003; 35: 545-54.
2 Dempfle CE. Coagulopathy of sepsis. Thromb Haemost 2004; 91: 213-24.
3 Sabroe I, Read RC, Whyte MK. et al. Toll-like receptors in health and disease: complex questions remain. J Immunol 2003; 171: 1630-5.
4 Akira S. Toll-like receptor signaling. J Biol Chem 2003; 278: 38105-8.
5 Bernard GR, Vincent JL, Laterre PF. et al. Recombinant human protein C Worldwide Evaluation in Severe Sepsis (PROWESS) study group. Efficacy and safety of recombinant human activated proteinC for severe sepsis. N Engl J Med 2001; 344: 699-709.
6 Gregory SA, Morrissey JH, Edgington TS. Regulation of tissue factor gene expression in the monocyte procoagulant response to endotoxin. Mol Cell Biol 1989; 09: 2752-5.
7 Osterud B. Tissue factor expression by monocytes: regulation and pathophysiological roles. Blood Coagul Fibrinolysis 1998; 09 (Suppl. 01) S9-14.
8 Osterud B, Flaegstad T. Increased tissue thromboplastin activity in monocytes of patients with meningococcal infection: related to an unfavourable prognosis. Thromb Haemost 1983; 49: 5-7.
9 Versteeg HH. Tissue factor as an evolutionary conserved cytokine receptor: Implications for inflammation and signal transduction. Semin Hematol 2004; 41 (Suppl. 01) 168-72.
10 Giesen PL, Rauch U, Bohrmann B. et al. Bloodborne tissue factor: another view of thrombosis. Proc Natl Acad Sci USA 1999; 96: 2311-5.
11 Bogdanov VY, Balasubramanian V, Hathcock J. et al. Alternatively spliced human tissue factor: a circulating, soluble and thrombogenic protein. Nat Med 2003; 09: 458-62.
12 Roca M, Martin-Comin J, Becker M. et al. A consensus protocol for white blood cells labelling with technetium-99m hexamethylpropylene amine oxime. EurJ Nucl Med 1998; 25: 797-9.
13 Rippmann JF, Pfizenmaier K, Mattes R. et al. Fusion of the tissue factor extracellular domain toa tumour stroma specific single-chain fragment variable antibody results in an antigen-specific coagulationpromoting molecule. Biochem J 2000; 349: 805-12.
14 Petersen LC. Active-site inhibited seven: mechanism of action including signal transduction. Semin Hematol 2001; 38: 39-42.
15 Hotchkiss RS, Swanson PE, Freeman BD. et al. Apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction. Crit Care Med 1999; 27: 1230-51.
16 Levi M, Ten Cate H. Disseminated intravascular coagulation. N Engl J Med 1999; 341: 586-92.
17 van Deventer SJ, Buller HR, ten Cate JW. et al. Experimental endotoxemia in humans: analysis of cytokine release and coagulation, fibrinolytic, and complement pathways. Blood 1990; 76: 2520-6.
18 Taylor Jr FB. Role of tissue factor and factor VIIa in the coagulant and inflammatory response to LD100 Escherichia coli in the baboon. Haemostasis 1996; 26 (Suppl. 01) 83-91.
19 Herwald H, Morgelin M, Bjorck L. Contact activation by pathogenic bacteria: a virulence mechanism contributing to the pathophysiology of sepsis. Scand J Infect Dis 2003; 35: 604-7.
20 Wuillemin WA, Fijnvandraat K, Derkx BH. et al. Activation of the intrinsic pathway of coagulation in children with meningococcal septic shock. Thromb Haemost 1995; 74: 1436-41.
21 Hartert H. Blutgerinnungsstudien mit der Thrombelastographie, einem neuen Untersuchungsverfahren. Klin Wschr 1948; 26: 577-83.
22 Salooja N, Perry DJ. Thrombelastography. Blood Coagul Fibrinolysis 2001; 12: 327-37.
23 Whitten CW, Greilich PE. Thromboelastography: past, present, and future. Anesthesiology 2000; 92: 1223-5.
24 Samama CM. Thromboelastography: the next step. Anesth Analg 2001; 92: 563-4.
25 Srinivasa V, Gilbertson LI, Bhavani-Shankar K. Thromboelastography: where is it and where is it heading?. Int Anesthesiol Clin 2001; 39: 35-49.
26 Swennen ELR, Bast A, Dagnelie PC. Immunoregulatory effects of adenosine 5´-triphosphate on cytokine release from stimulated whole blood. Eur J Immunol 2005; 35: 852-8.
27 Nooteboom A, van der Linden J, Hendriks T. Tumor necrosis factor-a and interleukin-1ß mediate endothelial permeability induced by lipopolysaccharide-stimulated whole blood. Crit Care Med 2002; 30: 2063-8.
28 Poeschl JMB, Leray C, Ruef P. et al. Endotoxin binding to erythrocyte membrane and erythrocyte deformability in human sepsis and in vitro . Crit Care Med 2003; 31: 924-8.
29 Muehlstedt SG, Richardson CJ, Lyte M. et al. Systemic and pulmonary effector cell function after injury. Crit Care Med 2002; 30: 1322-6.
30 Dehoux MS, Hernot S, Asehnoune K. et al. Cardiopulmonary bypass decreases cytokine production in lipopolysaccharide-stimulated whole blood cells: Roles of interleukin-10 and the extracorporeal circuit. Crit Care Med 2000; 28: 1721-7.
31 Rittersma SZ, Kremer JAHovinga, Koch KT. et al. Relationship between in vitro lipopolysaccharide-induced cytokine response in whole blood, angiographic in-stent restenosis, and TOLL-like receptor 4 gene polymorphisms. Clin Chem 2005; 51: 516-21.
32 Spolarics Z, Siddiqi M, Siegel JH. et al. Depressed interleukin-12-producing activity by monocytes correlates with adverse clinical course and a shift toward Th2-type lymphocyte pattern in severly injured male trauma patients. Crit Care Med 2003; 31: 1722-9.
33 Souter PJ, Thomas S, Hubbard AR. et al. Antithrombin inhibits lipopolysaccharide-induced tissue factor and interleukin-6 production by mononuclear cells, human umbilical vein endothelial cells, and whole blood. Crit Care Med 2001; 29: 134-9.
34 Chabry R. Lipopolysaccharide-binding molecules: transporters, blockers and sensors. Cell Mol Life Sci 2004; 61: 1697-1713.
35 Warren HS, Novitsky TJ, Ketchum PA. et al. Neutralization of bacterial lipopolysaccharides by human plasma. J Clin Microbiol 1985; 22: 590-5.
36 Opal SM, Scannon PJ, Vincent JL. et al. Relationship between plasma levels of lipopolysaccharide (LPS) and LPS-binding protein in patients with severe sepsis and septic shock. J Infect Disease 1999; 180: 1584-9.