A SIMPLIFIED AND LOW-COST ONE-STAGE CHROMOGENIC ASSAY FOR TISSUE FACTOR-DEPENDENT PROCOAGULANT ACTIVITY OF ENDOTHELIAL-CELLS

We developed a sensitive tissue factor (TF) chromogenic assay on a lim ited number of endothelial cells (EC), performed in microtiter plates, and which uses normal pooled human plasma instead of purified concent rates as a source of coagulation factors. Primary cultures of human um bilical vein EC (HUVEC), both unstimulated and stimulated by lipopolys accharide (LPS), were incubated with 50 mu l of diluted normal human p lasma (NHP) and 50 mu l of Factor Xa-specific chromogenic substrate (C BS 31-39, Stage, France). Hirudin was added at 4 U/ml to the plasma/CB S 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 purifi ed human Factor Xa. The following parameters were then defined :the nu mber of EC to plate (10(4) EC/well of a 96-well plate), the plasma-tes t dilution (1:20), the concentration of CBS 31-39 (0.50 mM) and the in cubation time of reagents with EC (2 hours). The procoagulant activity (PCA) measured was only dependent on TF since it was no longer detect able either when FVII-deficient plasma was tested instead of normal hu man plasma or when PCA assays were performed in the presence of a bloc king anti-human TF monoclonal antibody. This method allowed detection of a TF-dependent PCA on as few as 1000 EC per well. In addition, TF e xpression equal to 50% of maximal values was measured with LPS concent rations as low as 1 ng/ml, supporting the high sensitivity of the assa y.