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Endotoxin enhances the expression of monocyte prothrombinase activity
RA Robinson, L Worfolk and PB Tracy
Department of Biochemistry, University of Vermont College of Medicine,
Burlington 05405.
Thrombin is generated on the surface of mononuclear cells (MNCs) through
the assembly and function of the prothrombinase complex consisting of the
enzyme factor Xa, the cofactor/factor Va, calcium ions, and an appropriate
membrane surface for proper assembly of the protein constituents. Assays
performed in the presence of factors Va and Xa indicated that endotoxin
significantly enhanced the prothrombinase activity (1.5- to 2.5-fold; P
less than .001) expressed by MNCs in a dose- and time-dependent manner.
Monocytes present in the MNC suspensions were responsible for this
increased activity through processes resulting in both enhanced cellular
activity and the enhanced release of membranous vesicles. Endotoxin was
without effect on the expression of lymphocyte prothrombinase activity.
Scanning electron microscopy techniques indicated that endotoxin resulted
in extensive membrane blebbing of the monocytes present in the MNC
suspensions with no effect on the morphology of the lymphocytes. Within 5
hours, endotoxin maximally enhanced the prothrombinase activity expressed
by the monocyte membrane surface 2.8-fold, whereas 8 hours was required to
maximally enhance the activity associated with the released vesicles by
twofold. The observed increase in activity expressed by the monocyte
membrane surface was due solely to endotoxin, since the activity expressed
by the unstimulated monocyte membrane surface remained unaltered over time.
In contrast, cell vesiculation, which occurred in the absence of any
stimulus, was further enhanced by endotoxin. The increase in activity
associated with the released vesicles from both stimulated and unstimulated
cells paralleled an increase in the vesicle number as determined by flow
cytometric analyses. The vesicle released from both unstimulated and
stimulated monocytes were indistinguishable in size as determined by image
analysis and ranged between 0.05 and 0.3 microns in diameter.
2-Deoxy-D-glucose (2DG) significantly enhanced the prothrombinase activity
expressed by the monocyte membrane surface, as well as the released vesicle
fraction, when used alone or in addition to endotoxin. The enhanced
activity associated with the vesicle fraction again was attributed to the
release of more vesicles. In contrast, cycloheximide decreased the
prothrombinase activity expressed by the monocyte membrane surface, as well
as the activity associated with vesicles released from both stimulated and
unstimulated cells. These data suggest that the expression of monocyte
prothrombinase activity can be significantly enhanced by endotoxin through
processes that alter the monocyte membrane surface and augment the
vesiculation process. Both processes appear to be regulated by protein
synthesis and adenosine triphosphate (ATP)-dependent mechanisms.
Volume 79,
Issue 2,
pp. 406-416,
01/15/1992
Copyright © 1992 by The American Society of Hematology
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