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Prepublished online as a Blood First Edition Paper on May 1, 2003; DOI 10.1182/blood-2003-03-0826.
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Blood, 1 September 2003, Vol. 102, No. 5, pp. 1743-1744
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Brief report
Decreased factor VIII levels during acetaminophen-induced murine fulminant hepatic failure
Christopher B. Doering,
Ernest T. Parker,
Christopher E. Nichols, and
Pete Lollar
From the Winship Cancer Institute, Emory University, Atlanta, GA.
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Abstract
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During human fulminant hepatic failure (FHF) circulating levels of most
hemostatic proteins fall dramatically. Concurrently, factor VIII (fVIII)
procoagulant activity rises despite destruction of the hepatocytes considered
responsible for fVIII synthesis. This observation suggests a role for cells
other than hepatocytes in fVIII biosynthesis during FHF. We have attempted to
identify nonhepatocytic sites of fVIII biosynthesis by inducing FHF in mice
using acetaminophen overdose, a common cause of human FHF.
Acetaminophen-treated mice consistently displayed signs characteristic of FHF,
including elevated plasma aminotransferase activity. However,
acetaminophen-treated mice demonstrated markedly reduced fVIII activity,
contrary to the observation in human FHF. von Willebrand factor antigen levels
were only mildly reduced, suggesting that the decrease in fVIII is not
secondary to loss of von Willebrand factor. These results imply that there are
fundamental differences in the regulation of plasma fVIII levels between
humans and mice.
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Introduction
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Much of the knowledge concerning factor VIII (fVIII) biosynthesis has been
gained through the use of heterologous expression of fVIII transgenes in
cultured cells (reviewed in Kaufman et
al1). Unfortunately,
in vitro experiments cannot identify all of the factors involved in fVIII
biosynthesis in vivo. Thus, a substantial gap in the understanding of fVIII
gene regulation in vivo remains, including the identification of endogenous
sites of fVIII production. Beyond hepatocytes, several cell types in mice have
been shown to contain significant levels of fVIII
mRNA,2 but only
murine liver sinusoidal endothelial cells have been demonstrated to secrete
fVIII in
vitro.3,4
Additionally, liver is the only tissue that has been shown conclusively to
produce fVIII when transplanted into fVIII-deficient (hemophilia A) canines or
humans, as evident by alleviation of the bleeding
phenotype.5-7
However, during the course of fulminant hepatic failure (FHF) induced by
acetaminophen or viral hepatitis, plasma fVIII activity can increase to more
than 10 times normal
levels.8,9
The cellular source of this super-physiologic fVIII activity is currently
unknown. We previously reported that fVIII levels were decreased in a murine
model of FHF induced by the hepatotoxin,
azoxymethane.10
Because azoxymethane has not been proven to cause FHF in humans, we have
continued to identify other models that may be more representative of human
FHF pathophysiology. In the current study we sought to determine whether fVIII
levels are elevated in a recently described murine model of
acetaminophen-induced
FHF11 and possibly
identify alternative sites of fVIII biosynthesis in vivo.
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Study design
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Twelve-week-old C57BL/6J male mice (21-26 g) were purchased from Charles
River Laboratory (Wilmington, MA). Mice were fed standard rodent chow and were
kept on a 12-hour light and 12-hour dark cycle. Murine fulminant hepatic
failure was induced as described
previously.11
Briefly, mice were given an intraperitoneal injection of 100 mg/kg
phenobarbital (Sigma, St Louis, MO) suspended in corn oil, for 3 consecutive
days followed by an intraperitoneal injection of 250 mg/kg acetaminophen
(Sigma), dissolved in basic phosphate-buffered saline (10 mM
Na2HPO4, 150 mM NaCl, pH 11), on day 4. Twenty-four
hours after acetaminophen administration, the mice were killed, and blood was
collected by cardiac puncture into one-tenth volume 3.8% sterile trisodium
citrate. Plasma was isolated from whole blood by centrifugation at
1800g for 15 minutes at 4°C and stored at –70°C. The
control plasma used in this study was pooled plasma from 6 untreated C57BL/6J
male mice.
fVIII activity was determined using a chromogenic substrate assay in which
the rate of activation of factor X is linearly related to the concentration of
fVIII as follows. Plasma samples were diluted 1/81 in 20 mM HEPES
(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid),
0.15 M NaCl, 1% bovine serum albumen (BSA), pH 7.4. Thirty microliters of a
solution containing 3.6 nM factor IXa, 150 µM
phosphatidylcholine/phosphatidylserine vesicles (75%/25%), and 15 mM
CaCl2 was added to 30 µL diluted sample plasma. Next, 30 µL
of a solution containing 48 nM factor IIa and 900 nM factor X was added. After
the reaction mixture was incubated for 5 minutes at room temperature, 30 µL
of 1 mM Spectrozyme factor Xa (American Diagnostica, Stamford, CT), 400 nM
desulfatohirudin, 100 mM EDTA (ethylenediaminetetraacetic acid), 20 mM HEPES,
0.15 M NaCl, 0.1% polyethylene glycol, pH 7.4 was added, and the absorbance at
405 nm was measured over a 10-minute period. Murine plasma fVIII levels were
obtained by comparison to a standard curve using pooled normal human plasma
(FACT; George King Biomedical, Overland Park, KS). Aspartate aminotransferase
(AST) and alanine aminotransferase (ALT) activities were measured using the
Sigma Diagnostic Transaminases Reagent kit (Sigma) following the
manufacturer's instructions. von Willebrand factor (VWF) levels were measured
by enzyme-linked immunosorbent assay (ELISA) using a previously published
method.12 The ELISA
titer was defined as the absorbance at 490 nm obtained for a 1/20 dilution of
plasma. Data acquired at this dilution were within a linear range. All
numerical data are presented as means ± 1 standard deviation with the
data range presented in parentheses.
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Results and discussion
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Twenty-four hours after acetaminophen treatment, all mice displayed signs
of stage II hepatic encephalopathy, including reduced locomotive activity.
This treatment regimen previously has been shown to induce extensive hepatic
centrilobular necrosis within 24
hours.11 One
indicator of liver necrosis is the elevation of serum ALT activity and a
change in the AST/ALT ratio from greater than 1 to less than 1. Plasma from
control and acetaminophen-treated mice was assayed for AST and ALT activity
(Figure 1). Pooled plasma from
untreated mice had 30 U/mL AST activity and 19 U/mL ALT activity for an
AST/ALT ratio of 1.58, whereas the plasmas from all acetaminophen-treated mice
demonstrated elevated AST and ALT levels and decreased AST/ALT ratios. The
mean values for AST and ALT activity were 188 ± 10 (175-209) U/mL and
28 200 ± 6000 (20 300-37 300) U/mL, respectively, corresponding to an
AST/ALT ratio of 0.007. These findings indicate that FHF was successfully
induced in all of the mice.
Plasma fVIII activity was measured using a chromogenic substrate assay. The
concentration of fVIII activity measured in control mouse plasma was 2.9 U/mL,
which is consistent with previously published
results.13,14
Average fVIII activity was decreased by 91% to 0.27 ± 0.07 (0.16-0.37)
U/mL in the acetaminophen-treated murine plasmas
(Figure 2). Therefore, we were
unable to replicate the increase in plasma fVIII activity observed in human
acetaminophen-induced FHF in a murine model. The decrease in fVIII activity
observed in the present study corresponds well with the results previously
reported in azoxymethane-treated mice in which, at the highest dose of
azoxymethane tested (50 µg/g body weight), fVIII activity dropped to 10% of
control levels at 24 hours after
administration.10
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Figure 2.. FVIII activity versus VWF antigen levels in acetaminophen-treated
mice. Plasma from acetaminophen-treated mice (n = 12) was assayed for
fVIII activity by chromogenic assay and VWF antigen level by ELISA. Results
expressed are relative to pooled normal murine plasma.
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Several factors have been shown to influence circulating fVIII levels,
including VWF whose levels display a strong positive correlation with fVIII
levels (reviewed in Kamphuisen et
al15). Accordingly,
in the complete absence of VWF, fVIII levels are reduced by approximately 80%
in mice.12 We
measured VWF antigen levels in the acetaminophen-treated and control murine
plasmas and found that the levels in acetaminophen-treated mice were mildly
reduced to 62% ± 18% (29%-97%) of the control murine plasma level
(Figure 2). However, the extent
of decline in VWF was not equivalent to the decrease in fVIII, and there was
not a significant correlation between VWF antigen and fVIII activity within
the acetaminophen-treated mice. Therefore, the decrease in fVIII was not
secondary to a reduction in VWF. The current data support our previous
findings of reduced fVIII activity following azoxymethane-induced murine FHF.
Combined, these studies argue that there are fundamental differences in the
regulation of circulating fVIII levels between mice and humans and that these
differences should not be ignored when using the mouse as an experimental
model system to study fVIII biology.
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Footnotes
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Submitted March 18, 2003;
accepted April 22, 2003.
Prepublished online as Blood First Edition Paper, May 1, 2003; DOI
10.1182/blood-2003-03-0826.
Supported by National Institutes of Health grant R01-HL40921.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked "advertisement" in accordance with 18 U.S.C. section
1734.
Reprints: Pete Lollar, 1639 Pierce Dr, Rm 1003 Woodruff Memorial Bldg,
Emory University, Atlanta, GA 30322; e-mail:
jlollar{at}emory.edu.
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Top
Abstract
Introduction
)
and ALT (
) activity as described in "Study design." Error
bars represent 1 SD.