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Tissue factor pathway inhibitor and protease nexin-1 are major factor Xa
binding proteins on the HepG2 cell surface
Y Kazama, Y Komiyama and W Kisiel
Department of Pathology, University of New Mexico, School of Medicine,
Albuquerque 87131.
Previous studies indicated that human factor Xa bound to a human
hepatocellular carcinoma cell line (HepG2) that constitutively synthesizes
a factor V/Va molecule. Factor Xa binding to this cell line was not
measurably affected by pretreatment of the cells with anti- factor V IgG
and to a large extent (approximately 70%) was calcium- independent,
suggesting the presence of cell-surface binding proteins specific for
factor Xa other than factor V/Va. In the present study, we have further
characterized the interaction of factor Xa with the HepG2 cell and
performed chemical cross-linking and immunoprecipitation studies to
determine the identity of the HepG2 surface protein(s) interacting with
factor Xa. Initial studies demonstrated that HepG2- bound 125I-factor Xa
was not significantly displaced by unlabeled factor Xa blocked at the
active site with dansyl-L-glutamyl-glycyl-L- arginine (DEGR)-chloromethyl
ketone (DEGR-Xa), whereas DEGR-Xa effectively inhibited prothrombinase
activity of cell-bound factor Xa (Ki = 5 nmol/L). Essentially no
125I-DEGR-Xa binding to the HepG2 cells was observed, suggesting that an
intact factor Xa active site was a prerequisite for binding. 125I-factor Xa
binding to HepG2 cells was inhibited approximately 70% by pretreatment of
the cells with anti- tissue factor pathway inhibitor (TFPI) IgG in the
presence or absence of calcium ions, but was without effect on the
expression of prothrombinase activity. Immunoprecipitation of 125I-factor
Xa chemically cross-linked to its cell-surface binding protein with anti-
factor X IgG followed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) showed a complex with an apparent molecular
weight of 96,000. An identical molecular weight complex was observed
following immunoprecipitation of this radiolabeled complex with anti- TFPI
IgG. In addition to TFPI, approximately 30% of cell-bound factor Xa appears
to form a covalent complex with HepG2 cell-surface protease nexin-1 (PN-1)
as shown by pretreatment of the HepG2 cell with murine anti-PN-1 IgG. These
results suggest that approximately 1% to 2% of the factor Xa interacts with
HepG2 cell-surface factor V/Va to form a productive prothrombinase complex,
while the remaining factor Xa forms a non-productive complex with either
TFPI or PN-1.
Volume 81,
Issue 3,
pp. 676-682,
02/01/1993
Copyright © 1993 by The American Society of Hematology
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