Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 193-197
von Willebrand Factor Elevates Plasma Factor VIII Without
Induction of Factor VIII Messenger RNA in the Liver
By
Randal J. Kaufman,
Andrew J. Dorner, and
David N. Fass
From the Howard Hughes Medical Institute and the Department of
Biological Chemistry, University of Michigan, Ann Arbor, MI; the
Genetics Institute Inc, Andover, MA; and the Mayo Medical School,
Rochester, MN.
 |
ABSTRACT |
Factor VIII and von Willebrand factor (vWF) circulate in the plasma
as a noncovalent protein complex. Circulating levels of factor VIII are
coordinately regulated with circulating levels of vWF in which the
ratio is maintained at 1 molecule of factor VIII for 50 to 100 vWF
subunits. Infusion of vWF into vWF-deficient animal models and human
patients yields a secondary increase in circulating levels of factor
VIII. We have studied the mechanism of the secondary rise in factor
VIII in a porcine model of vWF deficiency. On infusion of vWF into a
vWF-deficient pig there was an approximately fivefold increase in
circulating factor VIII activity. Liver biopsies were taken pre- and
post-vWF infusion for isolation of total messenger RNA (mRNA). Factor
VIII-specific mRNA was measured by an RNAse protection assay. The
results showed no difference in the liver-specific factor VIII mRNA on
vWF infusion. These results indicate that the secondary rise in factor
VIII levels in response to exogenous vWF infusion is not dependent on
increased steady-state levels of factor VIII mRNA in the liver.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
FACTOR VIII IS THE X chromosome gene
product that is deficient or defective in the bleeding disorder
hemophilia A. Factor VIII functions in the intrinsic pathway of blood
coagulation as a cofactor to accelerate the activation of factor X by
factor IXa that occurs on a phospholipid surface in the presence of
calcium ions. The factor VIII amino acid sequence deduced from the
cloned complementary DNA (cDNA) identified that the molecule is
synthesized as a single-chain polypeptide having the domain structure
A1-A2-B-A3-C1-C21,2 and on secretion from the cell is
processed to a heterodimer consisting of a carboxy-terminal-derived
light chain of 80 kD in a metal ion-dependent association with a 200 kD amino-terminal-derived heavy-chain fragment.
In plasma, factor VIII is bound and stabilized by noncovalent
interactions with von Willebrand factor (vWF).3-8 In
addition, in vitro studies showed that vWF regulates factor VIII
activity through additional mechanisms: vWF prevents activation of
factor VIII by factor Xa9; vWF prevents inactivation of
factor VIII by activated protein C10-12; and vWF prevents
binding of factor VIII to phospholipids13,14 and to
thrombin-activated platelets.15 In addition, vWF can also
stimulate the stable accumulation of factor VIII on secretion from
transfected mammalian cells in culture.16,17 A primary
factor VIII binding site was identified within the amino-terminus of
mature vWF (residues 1 to 27218-20), and recent studies
suggest that both the amino-terminus and the carboxy-terminus of the
factor VIII light chain mediate interaction with vWF.21-25
Although each subunit of a vWF multimer contains one factor VIII
binding site, in vitro binding studies yielded conflicting data for
factor VIII:vWF subunit ratios of 1:1,26,27
1:4,28,29 and 1:10,15 to as low as 1:70
high-affinity binding sites.30 The source for the
difference remains unknown because different reagents, protein
concentrations, and assays were used for these studies. At least one
factor that influences this ratio is the conformation of vWF in the
assay.31 However, the ratio of circulating factor VIII to
vWF observed in vivo is tightly maintained at 1:50.7 Any
change in plasma vWF level is coupled with a concordant change in the
factor VIII level. The infusion of vWF into vWF-deficient animal models
and human patients immediately elevates factor VIII levels and the
factor VIII levels exhibit a sustained rise, even while vWF levels
decline as a result of clearance.3,4,7,8,32-34 In addition,
1-desamino-8-D-arginine vasopressin is used to treat bleeding episodes
in mild von Willebrand's disease (vWd) and mild hemophilia A and is
proposed to stimulate the endogenous release of vWF, thereby increasing
plasma levels of factor VIII.35 The mechanism by which vWF
stimulates factor VIII levels in plasma is not known. The presence of
vWF increases the plasma half-life of factor VIII from 2 to 3 hours to
12 to 14 hours.5,6 However, it is not known if vWF also
stimulates factor VIII synthesis as well. We have studied whether
plasma vWF levels influence factor VIII messenger RNA (mRNA)
expression.
A porcine model for severe homozygous vWF deficiency in humans exists.
In this porcine model of homozygous vWF deficiency the vWF antigen
level is 0.25% and the factor VIII activity is 15% to 30% of
normal.36 The genetic lesion responsible for the porcine
defect is thought to be a mutation outside of the coding region
influencing vWF mRNA stability.37 Infusion of vWF into the
circulation8 or perfusion of the porcine liver with
vWF38 elicits an increase in circulating factor VIII
activity. Thus, this model mimics the severe human vWF deficiency. In
this study, we have measured the effect of vWF infusion on the level of
factor VIII mRNA in the liver. The results show that infusion of vWF elicits a rise in circulating factor VIII at a post-transcriptional level.
| |
MATERIALS AND METHODS |
Infusion of vWF into vWF-deficient pig and liver biopsy.
Surgery was performed on a 35 kg female vWd pig 3 months and 3 weeks of
age. The animal was anesthetized intramuscularly with ketamine-xylazine-torbugesic (17.5 mg/kg, 2.5 mg/kg, and .025 mg/kg,
respectively) in divided doses. To provide vascular access, a Hickman
catheter was placed in the left carotid artery with the port tunneled
under the skin to the dorsal neck. The animal received 1 gm Ancef and
80 mg gentocin during catheter placement. A 5 cm incision below the
diaphragm to the right of the midline exposed the liver. A segment of
the margin of the liver was isolated using two Cooley aortic
clamps (Miltex Surgical Instruments, Lake Success, NY)
applied from opposite directions. This tissue was removed and placed
immediately into liquid nitrogen. An infusion of porcine vWF
concentrate was initiated to assist in hemostasis and to stimulate the
appearance of factor VIII in the circulation. While still secured with
the aorta clamps, the cut edge of the organ was sutured with No.
0 silk using a purse-string stitch. As the suture was drawn tight the
clamps were removed. Gelfoam was applied until bleeding stopped, the
liver was replaced, and the incision closed. Porcine vWF concentrate
was infused every 8 hours with a target plasma concentration of
50%. The animal was placed under constant human observation for the
following 99 hours (72 hours past the second procedure). After 27 hours, a second similar procedure was performed on the same animal
after it had received four infusions of porcine vWF totaling 3,000 U (ristocetin vWF activity). After the second biopsy, the animal was
provided additional infusions of porcine vWF three times a day to
prevent hemorrhage during recovery. At the time of the second biopsy
the factor VIII over-response was at twice the level of the ristocetin
vWF activity and five times the level of the vWF antigen measurements.
vWF preparation and vWF and factor VIII assay.
vWF concentrate was prepared by precipitation of porcine plasma with 1 mol/L potassium phosphate, pH 6.5, as described.39 The
precipitated proteins were suspended in approximately .08 plasma
volumes and dialyzed against 0.02 mol/L Na Citrate, 0.14 mol/L NaCl, pH
7.4. This procedure yields a product that contains on average 10 to 15 U/mL of ristocetin-vWF factor activity and 6 to 10 U/mL of vWF antigen.
Material prepared in this manner for infusion was frozen at
20°C for up to 5 months before its administration.
vWF-dependent, ristocetin-induced platelet agglutination was measured
using gel-filtered porcine platelets in the assay previously described.40 vWF antigen was determined by the method of
Laurell,41 using a rabbit antiporcine vWF antibody produced
in our laboratory.
Factor VIII activity was measured using a clotting assay with human
factor VIII-deficient plasma as substrate. In this modified activated
partial thromboplastin time assay normal pooled porcine plasma was used
as the standard. Units are therefore expressed as porcine units, with
100% (1 U/mL) being the activity found in the frozen (70°C)
porcine plasma pool.
Preparation of RNAse protection probe.
A HindIII-SmaI 441 base-pair fragment from the porcine
factor VIII B domain42 was subcloned into the
HindIII and SmaI sites of pGEM4. Plasmid DNA was
prepared and linearized by digestion with EcoRI restriction
endonuclease and 400 ng was used for in vitro transcription with
bacteriophage T7 RNA polymerase (Promega Biotech, Madison WI) in 50 mmol/L Tris-HCl pH 8.3, 75 mmol/L KCl, 3 mmol/L MgCl2, 10 mmol/L dithiothreitol, RNAsin (Promega Biotech, Madison WI), 100 µmol/L guanosine triphosphate (GTP), and 2 mmol/L of each nucleotide
adenosine triphosphate (ATP), cytidine triphosphate (CTP), and uridine
triphosphate (UTP), in the presence of 100 µmol/L Ci
[32P]-
GTP (New England Biolabs, Beverly, MA). After
incubation at 40°C for 1 hour, RQ1 DNase (Promega Biotech) was
added and incubated another 15 minutes at 37°C. The mixture was
extracted with phenol:chloroform (1:1) and 5 µg of yeast transfer RNA
(tRNA) was added for ethanol precipitation. After centrifugation, the
RNA pellet was dried and resuspended in diethylpyrocarbonate-treated
H2O.
Total RNA isolation and RNAse protection.
Total RNA was extracted from 0.2 gm of frozen porcine
liver biopsy samples. The frozen tissue was pulverized using a mortar and pestle, placed in guanidine thiocyanate, and homogenized in a
dounce homogenizer.43 The RNA was then purified over a
cesium chloride gradient.44 For control, RNA was isolated
from Chinese hamster ovary (CHO) cells as previously
described.45 Aliquots of RNA (10 µg and 15 µg) were
mixed with 1 × 106 cpm of 32P-labeled T7
transcript and dried down under vacuum. Samples were resuspended in
80% formamide, 40 mmol/L PIPES pH 6.4, 400 mmol/L NaCl, 1 mmol/L EDTA,
and heated to 85°C for 15 minutes. Samples were incubated at
45°C overnight. After incubation, samples were treated with
pancreatic ribonuclease (0.5 ug/mL) and RNAse T1 (50 ng/mL) in the
presence of 30 mmol/L Tris-HCl pH 8.0, 200 mmol/L NaCl, 100 mmol/L
LiCl, and 1 mmol/L EDTA for 30 minutes at 37°C. Reactions were
stopped by addition of sodium dodecylsulfate (SDS) to 0.5% and
proteinase K (130 µg/mL) and incubation 15 minutes at 37°C.
Samples were extracted with phenol:chloroform and ethanol precipitated
after addition of 5 µg yeast tRNA. Precipitated samples were
resuspended in formamide buffer for electrophoresis on a polyacrylamide
gel. Radioactive gels were exposed for autoradiography. Band
intensities were quantitated by NIH Image software (public domain).
Measure of poly(A)+ RNA.
A poly(T) probe was synthesized as first-strand cDNA by reverse
transcription of HeLa cell poly(A)+ RNA. A 100 µL
reaction contained 500 ng/µL poly(A)+ RNA, 10 mmol/L
dithiothreitol, 50 mmol/L Tris-HCl pH 8.3, 75 mmol/L KCl, 3 mmol/L
MgCl2, 0.5 U/µL RNAguard (Pharmacia, Piscataway, NJ), 1 mmol/L thymidine triphosphate (TTP),
10 µg/mL oligo (dT)12-18, 100 µCi -32P
dTTP (3000 Ci/mmol, Amersham, Arlington Heights, IL/US Biochemicals, Cleveland, OH), and 5 U/µL Moloney murine leukemia virus
reverse transcriptase (GIBCO-BRL, Gaithersburg, MD). The reaction was incubated at 42°C for 90 minutes and then terminated with the addition of 100 mmol/L NaOH and 10 mmol/L EDTA. The reaction was incubated at 65°C for 30 minutes to hydrolyze the
poly(A)+ template. The alkaline reaction was neutralized
with 100 mmol/L Tris-HCL, pH 7.5 for 15 minutes at room temperature.
The probe was separated from unincorporated radionucleotides by ethanol precipitation after addition of ammonium acetate to 0.3 mol/L. The
average length of the probe was 200 to 300 bases.
RNA samples were denatured with 500 µL of ice-cold 10 mmol/L NaOH and
1 mmol/L EDTA and were immediately applied to a nitrocellulose membrane
using a dot-blot apparatus. Wells were rinsed with 500 µL cold 10 mmol/L NaOH and 1 mmol/L EDTA and dried under vacuum. Filters were
rinsed in 2× SSC (150 mmol/L NaCl, 15 mmol/L sodium citrate) with
0.1% SDS and cross-linked using a GSG gene linker (Bio-Rad
Laboratories, Richmond, VA). Filters were pretreated and
hybridized with the poly(T) probe in 5× SSCPE (0.9 mol/L NaCl, 50 mmol/L Na2HPO4, 50 mmol/L EDTA) with 5×
Denhardt's solution,46 0.1% SDS, 100 µg/mL denatured
salmon sperm DNA, and 50% formamide. Band intensities were quantitated
by NIH Image software.
| |
RESULTS |
To measure the effect of vWF infusion on circulating levels of factor
VIII in the plasma and factor VIII mRNA levels in the liver, plasma
samples were taken from a homozygous vWF-deficient pig to obtain
baseline levels of factor VIII activity, vWF antigen, and vWF activity
levels. At 1 day and 4 days (not shown) before vWF infusion the levels
of vWF were measured for ristocetin cofactor activity and were not
detectable. The level of vWF antigen measured by quantitative
immunoelectrophoresis was also not detectable, although factor VIII
activity was detected at approximately 25% (Fig 1). Before infusion of vWF, a liver
biopsy was performed for extraction of RNA. Purified porcine vWF
(approximately 700 U) was infused into the homozygous vWF-deficient
pigs and subsequently every 8 hours. After infusion of porcine vWF,
plasma samples were obtained and monitored for factor VIII activity,
vWF antigen, and ristocetin cofactor activity (Fig 1). vWF levels,
measured by ristocetin cofactor activity, increased to approximately
30% immediately after infusion and with additional infusions,
subsequently increased over the next 24 hours up to approximately 60%
and vWF antigen up to 20%. These levels roughly correlated with
the ristocetin cofactor and vWF antigen determinations previously
reported for this form of porcine concentrate.47 Factor
VIII activity levels increased steadily over the 24-hour period
coincident when the vWF infusions were initiated and after 24 hours the
plasma factor VIII activity was 131%. At 27 hours postinfusion,
another liver biopsy was performed. After the second biopsy, continued
infusion of porcine vWF was maintained (2,100 ristocetin cofactor Units per 24 hours) to provide effective hemostasis. The elevation in factor
VIII activity in the plasma was sustained throughout the 72-hour period
after the second biopsy, a time during which the animal was under
constant human observation.
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| Fig 1.
Factor VIII and vWF levels pre- and post-vWF infusion.
Plasma samples were isolated at the indicated periods of time from a
vWF-deficient pig. The times that vWF infusions were performed are
indicated by the "I" arrows. vWF ristocetin cofactor activity and
antigen and factor VIII activity were measured as described in
Materials and Methods and presented as a percent of normal porcine
values. Liver biopsies were performed at the times indicated by the
"B" arrows. Factor VIII,  ; Ristocetin cofactor,  ; vWF
antigen,  .
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Factor VIII mRNA in liver biopsy samples was quantified by an RNAse
protection assay. The probe was an antisense RNA transcript containing
441 nucleotides of the porcine factor VIII B domain. The total length
of the purified probe was 471 nucleotides due to the presence of 30 nucleotides derived from the pGEM4 polylinker site. The vector-derived
sequences allowed the clear separation of undigested probe from the
RNAse protected fragment. Analysis by polyacrylamide gel
electrophoresis showed a protected fragment migrating at approximately
440 nucleotides in both RNA samples from liver biopsies isolated pre-
and post-vWF infusion (Fig 2A). Two
concentrations of RNA were used to confirm that the probe was in
excess. The protected fragment was not detected in RNA isolated from
CHO cells, showing specificity for the protection. This analysis did
not detect a significant difference in the amount of protected fragment
observed in RNA isolated before vWF infusion compared with RNA isolated
27 hours after vWF infusion. RNA dot blot analysis of the same samples
using a poly(T) probe (as a measure of poly(A)+ mRNA)
indicated that the inability to detect a difference in factor VIII mRNA
was not due to differences in the amount of poly(A)+
containing RNA (Fig 2B). Hybridization to poly(T) was not detected with
yeast tRNA, showing specificity of the hybridization. At the time point
of RNA analysis, serum levels of factor VIII were significantly
elevated. This result indicates that factor VIII mRNA levels were not
elevated at this time point in the liver.
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| Fig 2.
Factor VIII mRNA levels in the liver pre- and post-vWF
infusion. Total RNA was isolated from samples of porcine liver biopsied
pre- and post-vWF infusion. The total RNA samples (10 and 15 mg) were
assayed by RNAse protection as described in Materials and Methods
(Panel A). Analysis of a 1/50 aliquot of the probe (lane 1) showed that
under these assay conditions the radiolabeled probe was in vast excess.
Densitometry of the autoradiogram indicated that levels of factor VIII
mRNA did not significantly differ between pre-vWF infusion (relative
band intensities were: 1.0 U/10 mg RNA and 1.8 U/15 mg RNA) and
post-vWF infusion (1.1 U/10 mg RNA and 2.0 U/15 mg RNA). The amount of
poly(A)+ RNA was determined by dot-blot hybridization to
a poly(T) probe (panel B). For controls total CHO cell RNA and yeast
tRNA were analyzed as indicated. Densitometry of the dot-blot showed
that pre- (area = 4.5) and post-vWF (area = 4.8) infusion
samples did not significantly differ in their hybridization to the
poly(T) probe.
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| |
DISCUSSION |
The observation that hemophilia A offers protection from ischemic heart
disease48 and evidence that elevated factor VIII is
associated with thrombotic disease49 provides an incentive to understand the mechanism by which factor VIII levels are regulated in plasma. It has long been known that plasma levels of vWF can regulate factor VIII levels. At least one mechanism involves
stabilization of factor VIII in the blood.5,6 However, on
infusion of vWF into vWF-deficient patients, factor VIII levels exhibit
a sustained increase, even in the presence of decreasing levels of vWF
due to its clearance.33,34 These observations prompted us
to evaluate whether factor VIII mRNA expression is also regulated by
circulating levels of vWF. Our studies did not detect any increase in
factor VIII-specific mRNA in the liver, although circulating levels of factor VIII in the plasma were greatly elevated. Therefore, if vWF
elicits an increase in factor VIII synthesis, it does not exert this
effect through a change in the steady-state level of factor VIII mRNA
in the liver.
To date, most evidence supports that factor VIII is synthesized in the
liver. Factor VIII mRNA and protein is detected in many tissues
including liver, spleen, lymph node, kidney, and placental
extracts.50-53 Transplantation of liver as well as
reticuloendothelial tissue have successfully sustained physiologically
functional levels of factor VIII in the plasma.54-59
Therefore, we cannot rule out that factor VIII mRNA levels do rise in
tissues other than the liver in response to vWF infusion.
Our findings support that vWF regulates factor VIII levels by
protecting factor VIII from clearance and also possibly by stabilizing factor VIII on secretion from the site of synthesis. The results of
these studies suggest that the regulation of factor VIII transcription in the liver is not a contributing factor in the regulation of plasma
factor VIII levels by vWF. Therefore, strategies for liver-directed gene therapy that use promoter elements other than factor VIII, would
be justified if the expression vector provides sufficient level of
factor VIII in the plasma. Because vWF appears to be a significant
factor in the regulation of factor VIII plasma levels, it is possible
that an increase in the synthesis of factor VIII above normal may not
result in excess factor VIII in the plasma because vWF binding would be
limiting. Further studies are required to directly test this
hypothesis. If this turns out to be true, then a thrombotic concern for
the overexpression of factor VIII mRNA in gene therapy may be
minimized.
| |
ACKNOWLEDGMENT |
We thank Mariot Varban, Micheline Moussalli, Joseph Nowak, and Luigina
Tagliavacca for technical assistance and Steven Pipe for critical
comments on the manuscript.
| |
FOOTNOTES |
Submitted February 19, 1998;
accepted September 4, 1998.
Supported by National Institutes of Health grant HL52173 (R.J.K.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address correspondence to Randal J. Kaufman, Howard Hughes Medical
Institute and the Department of Biological Chemistry, University of
Michigan, 4570 MSRB 11, 1150 W. Medical Center Dr, Ann Arbor, MI 48109;
e-mail:kaufmanr{at}umich.edu.
| |
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Abstract
Introduction
