The effect of cultured endothelial cells on factor VIII procoagulant
activity
NW Stead and PA McKee
Cultured human umbilical vein endothelial cells produce a protein that has
von Willebrand factor activity and forms immunoprecipitates with rabbit
antibody to purified plasma factor VIII/von Willebrand factor (FVIII/vWF)
protein, but it has no FVIII procoagulant activity. Of the three
characteristics of plasma FVIII/vWF protein, only FVIII procoagulant
activity is readily destroyed by trace proteases. A previous report from
this laboratory demonstrated protease activity in culture medium under
conditions that had been used by others to show that endothelial cells do
not synthesize protein with FVIII procoagulant activity. However, even if
cultured endothelial cells are placed in protease-free culture medium, no
FVIII procoagulant activity can be detected, despite an increase in the
level of protein with vWF activity from 0 to 0.57 microgram/ml by 48 hr.
This observation and the lack of protease activity in medium left in
contact with the cells for 48 hr led to the hypothesis that proteases exist
on the surface of cultured umbilical vein endothelial cells. Protease
activity was quantitated by the hydrolysis of p-nitroaniline from the
substrate, N- benzoyl-phenylalanyl-valyl-arginyl-p-nitroanilide and by
degradation of the procoagulant activity of added purified plasma FVIII/vWF
protein. In the absence of endothelial cells, no protease activity was
present in protease-free culture medium whether or not it had previously
overlaid cultured cells. This medium did not cause cleavage of p-
nitroaniline from the tripeptide substrate, and 83% of added FVIII
procoagulant activity remained after 48 hr. When the synthetic tripeptide
was incubated in contact with cultured endothelial cells, 7.3 +/- 0.8 X
10(-10) moles of p-nitroaniline/hr was released; moreover, only 47% of the
added FVIII procoagulant activity remained after 48 hr. Given this rate of
destruction, it can be calculated that sufficient protease activity exists
on the surface of cultured endothelial cells to degrade the procoagulant
activity of approximately 1.6 microgram FVIII/vWF protein/hr. This
degradation rate is 45 times the rate of release of FVIII/vWF protein from
cultured endothelial cells when assessed by the generation of protein with
vWF activity. Hence, the detection of FVIII procoagulant activity, if in
fact synthesized by cultured endothelial cells, will be most difficult.
Volume 54,
Issue 3,
pp. 560-572,
09/01/1979
Copyright © 1979 by The American Society of Hematology
