Thrombelastography for the monitoring of lipopolysaccharide induced activation of coagulation

Abstract

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.

Endotoxinemia-induced changes in coagulation as measured by rotation thrombelastometry technique and conventional laboratory tests: results of a pilot study on pigs

“…Following this mild exposure to endotoxin, a significantly shortened CT was noted, whereas CFT and MCF were not affected. Similar results were reported in an in-vitro study [12]. Most likely, these somewhat different results refer to the different thrombelastographic techniques and dosages and kinds of lipopolysaccharide used.…”

“…ROTEM, using activated tests, correlates highly with classical thrombelastography methods under similar circumstances [6,10]. However, reports on ROTEM monitoring of the coagulation system for inflammation, severe sepsis or septic shock are rare in the literature [11][12][13]. To test the hypothesis that the ROTEM technique allows detection of endotoxinemiainduced changes in hemostasis, we conducted a pilot study on pigs and compared the results of ROTEM assays with those of conventional coagulation tests recommended for diagnosing DIC.…”

Thromboelastometry (TEM®) Findings in Disseminated Intravascular Coagulation in a Pig Model of Endotoxinemia

“…Thus, increased LPS-induced activation of the coagulation process was not mirrored at the early stage by the routine coagulation test results. A similar CT reduction in an in vitro model was reported by Zacharowski et al (22), who incubated whole blood with different concentrations of LPS (0.005-1 mg/mL) for 4 h. Increasing amounts of LPS resulted in a substantial shortening in CT, and the half-maximal effective concentration (ED50) of LPS effect on CT was recorded at 18 μg/mL (22). The same study also showed that ex vivo addition of 10 ng/mL LPS to platelet-poor plasma did not change CT.…”

Changes in fibrinolysis and severity of organ failure in sepsis: A prospective observational study using point-of-care test—ROTEM

“…To preserve physiological conditions and to quantify intrinsic changes in the tissue factor triggered coagulation, no activators were added to the ROTEM system, similar to 2 previous studies [11,13]; furthermore, in vitro experiments have shown that endotoxin-initiated clotting activation is not accurately measured by thromboelastometry when additional clotting activators are used [23]. Plasminogen activator inhibitor-1 is the main inhibitor of fibrinolysis in sepsis and has several conformations including its inactive and latent forms [24].…”