Blood, Vol. 95 No. 4 (February 15), 2000:
pp. 1465-1472
Extracellular granzyme A, complexed to proteoglycans, is protected
against inactivation by protease inhibitors
Elisabeth H. A. Spaeny-Dekking,
Angela M. Kamp,
Christopher J. Froelich, and
C. Erik Hack
From the CLB, Sanquin Blood Supply Foundation, Department of
Pathophysiology Plasma Proteins, and Laboratory for Experimental and
Clinical Immunology, Academic Medical Center, University of Amsterdam,
The Netherlands; the Department of Research, Evanston Hospital,
Northwestern University, Evanston, IL; and the Department of Internal
Medicine, Free University Hospital, Amsterdam.
Granzyme A (GrA) and B (GrB) together with perforin are the main
constituents of cytotoxic granules of cytotoxic T lymphocytes (CTLs)
and natural killer (NK) cells. The cytotoxic proteins are released to
deliver a lethal hit during contact between the CTL or NK cell and
target cell. With the use of an enzyme-linked immunosorbent assay for
antigenic levels, we showed in a recent study that plasma of patients
with activated CTLs and NK cells contain elevated levels of
extracellular GrA. In this study, we determined the form and
proteolytic capacity of this extracellular GrA detected in plasma. With
the use of various assays, we show that part of the extracellular GrA
circulates in the mature conformation and is bound to proteoglycans
that protect it against inactivation by protease inhibitors, such as
antithrombin III and 
-2-macroglobulin, whereas another part of GrA
circulates as a complex with antithrombin III. Finally, with the use of
a novel assay for active GrA, we demonstrate that some plasma samples
with high levels of extracellular GrA contain active GrA. These results
suggest that various forms of extracellular GrA occur in vivo and that
the regulation of GrA activity may be modified by proteoglycans. These
data support the notion that granzymes may exert extracellular
functions distant from the site of CTL or NK cell interaction with
their target cells.
