R. Consonni et Rm. Bertina, ANTIGENIC AND FUNCTIONAL EXPRESSION OF TISSUE FACTOR IN ENDOTOXIN-STIMULATED U937 CELLS - REGULATION OF ACTIVITY BY CALCIUM IONOPHORE A23187, Thrombosis and haemostasis, 74(3), 1995, pp. 904-909
Tissue Factor (TF) is a transmembrane glycoprotein that serves as cofa
ctor for Factor VII (FVII) in the initiation of blood coagulation and
that is differentially expressed in a number of cell types. being cons
titutively expressed in some and inducible in others. We studied the l
ocalization and the functional activity of TF in monocytic leukemia U9
37 cells at different rime intervals after lipopolysaccharides (LPS) s
timulation, and the effect of calcium ionophore on the surface express
ed TF. Exposure of U937 cells to 10 mu g/ml LPS resulted in a time dep
endent increase of TF expression that reached a maximum at 12 h for TF
antigen and at 24 h for TF activity. Blocking of surface TF with inhi
bitory anti-TF antibody abolished >93% of the activity of lysed cells
stimulated for 24 h, while it blocked only 80% of the activity in lyse
d cells stimulated for 12 h suggesting that at that time about 20% of
TF is not accessible for the antibody. Even at 24 h when the specific
activity of surface expressed TF is 5.5 times higher than at 12 h, thi
s specific activity is still 10 fold lower than that of TF in lysed ce
lls. Addition of Ca++ ionophore A23187 to LPS stimulated cells resulte
d in a fast increase of TF activity that was dependent on the dose of
ionophore, on the extracellular Ca++ concentration and on the time tha
t the cells had been incubated with LPS. Kinetic analysis of Factor X
hydrolysis by the TF/FVII complex demonstrated that the addition of io
nophore resulted in an increase of both the Vmax and the apparent Km,
however, without affecting the catalytic efficiency of the reaction. T
hese data suggest that in U937 cells part of the TF induced by LPS is
cryptic, that the specific activity of surface expressed tissue factor
increases with time and that intracellular changes in Ca++ concentrat
ion can be important in the regulation of the expression of TF activit
y on the cell surface.