Validation of a novel tissue factor assay in experimental human endotoxemia.
BACKGROUND
Nuclear factor kappa B (NF-kappa B) activation and tissue factor (TF) expression may contribute to lethality in sepsis. Inappropriate in vivo expression of TF is likely responsible for fibrin deposition in sepsis-associated disseminated intravascular coagulation (DIC). Clinical assessment of TF expression has remained a major challenge. No point-of-care assays are currently available to measure the level of TF activity in whole blood. The current study examined the suitability of the TiFaCT assay as a point-of-care assay to detect TF expression.


METHODS
30 healthy male volunteers received 2 ng/kg of LPS. Tissue factor-dependent coagulation was quantified with a novel assay called tissue factor clotting time (TiFaCT), and by measurement of activation markers of downstream coagulation.


RESULTS
Ex vivo addition of anti-TF antibodies to blood slightly increased clotting times at 0-24 h (p<0.01) indicating that some tissue factor activity was present in whole blood at any time. LPS bolus infusion decreased TiFaCT clotting time by -23% compared to baseline (p<0.01), when in vivo clotting increased, as demonstrated by a 10-fold increase in prothrombin fragment levels (F1+2). Ex vivo incubation with LPS considerably shortened TiFaCT (from 1000s to 400s as compared to control incubation; p<0.01). This effect was blunted at 2-4 h after LPS infusion (i.e. the time of monocytopenia), but twofold enhanced 24 h after LPS challenge (p<0.01).


CONCLUSIONS
In summary, the TiFaCT assay was validated in our in vivo model of LPS-induced coagulation. It detected minute quantities of circulating TF even at baseline. TiFaCT is shortened at times of in vivo thrombin generation.
Marsik
Claudia
C
Department of Clinical Pharmacology, School of Medicine and Institute for Medical and Chemical Laboratory Diagnostics, Vienna University, Waehringer Guertel 18-20, A-1090 Vienna, Austria.
Quehenberger
Peter
P
Mackman
Nigel
N
Osterud
Bjarne
B
Luther
Thomas
T
Jilma
Bernd
B
D000328:Adult; D001774:Blood Chemical Analysis / Q000379:methods*; D001780:Blood Coagulation Tests / Q000379:methods*; D019446:Endotoxemia / Q000097:blood* / Q000139:chemically induced; D004731:Endotoxins; D006801:Humans; D008297:Male; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013925:Thromboplastin / Q000032:analysis*
D004731:Endotoxins; D013925:Thromboplastin