Thrombelastography for the monitoring of lipopolysaccharide induced activation of coagulation.
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 incubation periods (0-5 hrs), thereafter rotation thrombelastography was performed. Incubation of whole blood (>or=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 alpha, clot formation time, maximal clot formation) remained unaltered. EC(50) of the LPS effect on whole blood clotting time was 18 microg/ml. In isolated leukocytes, 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 be a sensitive and reliable tool for the determination of LPS-induced tissue factor mediated activation of haemostasis in whole blood samples.
Zacharowski
Kai
K
Department of Anesthesiology, University Hospital Dusseldorf, Moorenstrasse 5, D-40225 Dusseldorf, Germany.
Sucker
Christoph
C
Zacharowski
Paula
P
Hartmann
Matthias
M
D001777:Blood Coagulation / Q000187:drug effects*; D003513:Cycloheximide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D066298:In Vitro Techniques; D007962:Leukocytes / Q000187:drug effects / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D015203:Reproducibility of Results; D013916:Thrombelastography / Q000379:methods; D013925:Thromboplastin / Q000378:metabolism / Q000494:pharmacology; D014914:Whole Blood Coagulation Time
D008070:Lipopolysaccharides; D011500:Protein Synthesis Inhibitors; D013925:Thromboplastin; D003513:Cycloheximide