A simplified and low-cost one-stage chromogenic assay for tissue factor dependent procoagulant activity of endothelial cells

Thrombosis Research
C PouplardY Gruel

Abstract

We developed a sensitive tissue factor (TF) chromogenic assay on a limited number of endothelial cells (EC), performed in microtiter plates, and which uses a normal pooled human plasma instead of purified concentrates as a source of coagulation factors. Primary cultures of human umbilical vein EC (HUVEC), both unstimulated and stimulated by lipopolysaccharide (LPS) were incubated with 50 microliters of of diluted normal human plasma (NHP) and 50 microliters of Factor Xa-specific chromogenic substrate (CBS 31-39, Stago, France). Hirudin was added at 4 U/ml to the plasma/CBS 31-39 mixture to inhibit thrombin generation. Optical densities were read at 405 nm and corresponding amounts of generated factor Xa were expressed in mU Xa/well using a standard curve established with purified human Factor Xa. The following parameters were then defined: the number of EC to plate (10(4) EC/well of a 96-well plate), the plasma-test dilution (1:20), the concentration of CBS 31-39 (0.50 mM) and the incubation time of reagents with EC (2 hours). The procoagulant activity (PCA) measured was only dependent on TF since it was no longer detectable either when FVII-deficient plasma was tested instead of normal human plasma or when PCA assays were perf...Continue Reading

References

Nov 1, 1992·Cancer Metastasis Reviews·L V Rao
May 1, 1992·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·A MantovaniE Dejana
Jan 1, 1991·Haemostasis·F MarkwardtE Bucha
May 1, 1986·Proceedings of the National Academy of Sciences of the United States of America·P P NawrothD M Stern

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Citations

Dec 16, 1998·The Journal of Laboratory and Clinical Medicine·P Reverdiau-MoalicY Gruel
Mar 1, 2007·Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology·Robert H VonderheideScott J Antonia
Apr 5, 2001·American Journal of Physiology. Endocrinology and Metabolism·M P WautierJ L Wautier

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