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High-molecular weight kininogen is present in cultured human endothelial
cells: localization, isolation, and characterization
F van Iwaarden, PG de Groot, JJ Sixma, M Berrettini and BN Bouma
Department of Haematology, University Hospital Utrecht, The Netherlands.
The presence of high-molecular weight (mol wt) kininogen was demonstrated
in cultured human endothelial cells derived from the umbilical cord by
immunofluorescence techniques. Cultured human endothelial cells contain 58
+/- 11 ng (n = 16) high-mol wt kininogen/10(6) cells as determined by an
enzyme-linked immunosorbent assay (ELISA) specific for high-mol wt
kininogen. High-mol wt kininogen was isolated from cultured human
endothelial cells by immunoaffinity chromatography. Nonreduced sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated
that endothelial cell high-mol wt kininogen consisted of five protein bands
with mol wts of 95,000, 85,000, 65,000, 46,000, and 30,000 daltons.
Immunoblotting of the endothelial cell high-mol wt kininogen by using
specific antisera against the heavy and light chain indicated that the
95,000-, 85,000-, and 65,000-dalton bands consisted of the heavy and light
chain whereas the 46,000- and 30,000-dalton bands reacted only with the
anti-light chain antiserum. Immunoprecipitation studies performed with
lysed, metabolically labeled endothelial cells and monospecific antisera
directed against high-mol wt kininogen suggested that high-mol wt kininogen
is not synthesized by the endothelial cells. Endothelial cells cultured in
high-mol wt kininogen-free medium did not contain high-mol wt kininogen.
These studies indicate that endothelial cell high-mol wt kininogen was
proteolytically cleaved in the culture medium and subsequently internalized
by the endothelial cells. Binding and internalization studies performed
with 125I-labeled, proteolytically cleaved, high-mol wt kininogen showed
that endothelial cells can indeed bind and internalize proteolytically
cleaved high-mol wt kininogen in a specific and saturable way.
Volume 71,
Issue 5,
pp. 1268-1276,
05/01/1988
Copyright © 1988 by The American Society of Hematology
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