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Human neutrophils synthesize thrombomodulin that does not promote
thrombin-dependent protein C activation
EM Conway, B Nowakowski and M Steiner-Mosonyi
Department of Medicine, Toronto Hospital, University of Toronto, Ontario,
Canada.
Thrombomodulin (TM) is a surface glycoprotein that forms a 1:1 complex with
thrombin, thereby interacting to form the basis of a major physiologically
relevant natural anticoagulant mechanism. Although initially described as a
vascular endothelial cell receptor, TM has been reported to be present in
several other cells, including megakaryocytes, platelets, monocytes, and
several cultured cells. Other investigators have reported that neutrophils
(PMN) may play a role in the hemostatic mechanism by supporting
transformation of prothrombin to thrombin. To determine whether PMN might
contribute further to the regulation of the coagulation system, we have
evaluated these cells for the expression of TM. Large numbers of human
leukocytes were isolated by standard techniques, and the PMN fraction was
extracted and shown to be free of platelets and monocytes. Membrane
preparations were affinity purified on an anti-TM-Affigel-10 matrix and the
eluted material was examined by Western blotting, sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, and silver staining. The
purified material was identical in apparent molecular weight to TM from
human placenta and human umbilical vein endothelial cells (HUVEC). Using a
sensitive and specific immunoassay, we estimated that there are a minimum
of 5,220 +/- 1,658 molecules of TM per PMN, as compared with more than
50,000 in HUVEC. Northern analysis of RNA from PMN indicates that specific
messenger RNA for TM, as identified by a single 3.8-kb band, is identical
to that from HUVEC, and thereby confirms that PMN can also synthesize the
receptor. Localization of TM in PMN was attempted by immunofluorescence,
and the receptor was visualized only in permeabilized PMN, but was not seen
on the surface of nonpermeabilized cells. Flow cytometry was also used, and
could detect TM in 10% to 15% of nonpermeabilized PMN, whereas the antigen
was present in greater than 80% of permeabilized cells. Biologic function
of the PMN-derived TM, as tested by thrombin-dependent activation of
protein C, was absent. Our results suggest that TM is synthesized by PMN,
but under nonstimulated conditions, the protein is largely excluded from
the membrane surface, and lacks the ability to promote activation of
protein C by thrombin. TM from PMN may provide a further link between
inflammation and thrombosis and may also be a significant source of plasma
TM.
Volume 80,
Issue 5,
pp. 1254-1263,
09/01/1992
Copyright © 1992 by The American Society of Hematology
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