From the Hemostasis and Thrombosis Research Center,
Department of Hematology, University Hospital, 2300 RC Leiden, The
Netherlands,
Hormone and Metabolic Research Unit, Louvain
University Medical School and Christian de Duve Institute of Cellular
Pathology, 1200 Brussels, Belgium, and ¶¶ Laboratory for
Experimental Internal Medicine, Academic Medical Center, 1105 AZ
Amsterdam, The Netherlands
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ABSTRACT |
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Protein C is a vitamin K-dependent
zymogen of a serine protease that inhibits blood coagulation by
proteolytic inactivation of factors Va and VIIIa. Individuals affected
by protein C deficiency are at risk for venous thrombosis. One such
affected individual was shown earlier to carry a 14 T
C mutation
in the promoter region of the protein C gene. It is shown here that the
region around this mutation corresponds to a binding site for the
transcription factor hepatocyte nuclear factor (HNF)-6 and that this
site completely overlaps an HNF-1 binding site. HNF-6 and HNF-1 bound
in a mutually exclusive manner. The
14 T
C mutation reduced HNF-6
binding. In transient transfection experiments, HNF-6 transactivated
the wild-type protein C promoter and introduction of the mutation abolished transactivation by HNF-6. Similar experiments showed that
wild-type protein C promoter activity was reduced by cotransfection of
an HNF-1 expression vector. This inhibiting effect of HNF-1 was
reversed to a stimulatory effect when promoter sequences either upstream or downstream of the HNF-6/HNF-1 site were deleted. It is
concluded that HNF-6 is a major determinant of protein C gene activity.
Moreover, this is the first report describing the putative involvement
of HNF-6 and of an HNF-6 binding site in human pathology.
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INTRODUCTION |
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Protein C, which is a vitamin K-dependent zymogen of a serine protease synthesized in the liver, plays an important role in the regulation of blood coagulation. Activated protein C functions as an anticoagulant by inactivating two of the regulatory proteins of the coagulation pathway, factors Va and VIIIa (1, 2). Furthermore, activated protein C stimulates fibrinolysis through the neutralization of plasminogen activator inhibitor-1 (3). The physiological significance of the anticoagulant activity of protein C is shown in individuals homozygous or compound heterozygous for protein C deficiency. These individuals suffer from massive disseminated intravascular coagulation or neonatal purpura fulminans (4-6). Individuals affected by heterozygous protein C deficiency, although more mildly affected, are at risk of thrombophlebitis, deep vein thrombosis, or pulmonary embolism (7-9).
Transcription of eukaryotic genes by RNA polymerase II involves DNA elements located within the promoter region and transcription factors that associate with these DNA elements (10). Certain transcription factors, such as the TATA box-binding protein TBP, specify the transcription initiation site (11), whereas others regulate the efficiency of transcription initiation. This latter group of transcription factors comprises both ubiquitous and tissue-specific factors. Protein C promoter activity is liver-specific and controlled by liver-specific and by ubiquitous transcription factors. Binding sites have been identified in this promoter for the liver-enriched factors hepatocyte nuclear factor (HNF)1-1 and HNF-3 and for the ubiquitous factor NF-1, and the activity of these factors was shown to be synergistic (12-16).
Genetic analysis of individuals suffering from hereditary protein C
deficiency suggested that HNF-1 (13-15) and HNF-3 (12, 14, 15) are
involved in protein C gene transcription, by binding to nucleotides
10 to
22 and
26 to
37 and
33 to
22, respectively. HNF-1
binding to and HNF-1 transactivation of the protein C promoter was
abolished in a promoter construct where the naturally occurring
14 T
C mutation (13) had been introduced. This mutation is associated
with type I protein C deficiency (13).
It is shown here that this HNF-1 binding site also binds HNF-6, a recently cloned liver-enriched transcription factor (17). HNF-6 contains a bipartite DNA binding region consisting of a novel type of homeodomain and of a single cut domain (17). In the liver, HNF-6 controls the activity of genes that code for plasma carrier proteins and for enzymes regulating glucose metabolism (17). This work shows that HNF-6 stimulates the protein C gene promoter and that a mutation in this promoter, which is associated with protein C deficiency in patients, leads to a loss of HNF-6 binding and activity. Finally, promoter sequences located both upstream and downstream of the HNF-6/HNF-1 binding site are important for transactivation by both HNF-6 and HNF-1.
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EXPERIMENTAL PROCEDURES |
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Polymerase Chain Reaction (PCR) Amplification--
Fragments of
the human protein C promoter region were PCR-amplified, using pCwtCAT
(12) as template, with different combinations of the following
oligonucleotides:
5'-CAGCGTCCCCGGGCTTGTATGGTGGCACATAAATACATGT-3' (5' pr1,
396 to
357, all nucleotide numbering is relative to the
transcription start site) (18);
5'-TGAGACCACATCTGTCCCGGGTTTTG-3' (5'pr2,
202 to
177);
5'-TTGCCCTCACCTCCCTCCCGGGTGGAT-3' (5'pr3,
179
to
153); 5'-GGCAGACCCGGGCTTCGGGCAGAACAAG-3' (5'pr4,
139 to
112); 5'-TAGGACCAGGAGTGCCCGGGCCACT-3'
(5'pr5,
100 to
76); 5'-CAGCGTCCCCGGGCTTGTATGGTGGCACATAAATACATGT-3' (5'pr6,
396 to
357); 5'-TAACTCGAGCTCCAGGCTGTCATG-3' (5'pr7,
13 to +13); 5'-CTCTTCTCTTCTCCCGGGGGCAGCCCTCCCTCCACACCCCTCATA-3' (3'pr1, +122 to +78); 5'-TCACACCCGGGATAGACCTGCCTGGA-3'
(3'pr2, +91 to +65); 5'-TCACAGGCCCGGGTCGTGGAGATAC-3'
(3'pr3, +58 to +34); 5'-TGCTTGGAGCTCAGCACTGAGGCCT-3'
(3'pr4,
33 to
57) and
5'-CTCTTCTCTTCTCCCGGGGGCAGCCTCCCTCCACACCCTCATA-3' (3'pr5, +122 to
+78). The nucleotides underlined in the sequences are not present in
the protein C promoter region and introduce SmaI or
Eco1CRI sites in the PCR fragments. Amplifications were performed in a 50-µl reaction mixture containing 10 mM
Tris-Cl, pH 8.0, 1 mM MgCl2, 50 mM
KCl, 350 ng of primers, 100 ng of genomic DNA, 250 µM
dNTPs, 60 mg/ml bovine serum albumin, and 0.3 unit of Taq
polymerase. After an initial incubation at 91 °C for 4 min, 32 cycles were carried out at 91 °C for 1 min, 56 °C for 1 min, and
72 °C for 1 min.
Plasmid Constructions--
The pCAT00 vector (19) was used to
make reporter constructs containing protein C promoter sequences
driving chloramphenicol acetyltransferase (CAT) gene expression.
PCR-amplified DNA fragments were digested with SmaI and
cloned into the SmaI site of pCAT00. The reporter constructs
were named to indicate the length of the promoter fragment as follows:
pCCAT493 (5'pr1/3'pr1), pCCAT291 (5'pr2/3'pr1), pCCAT267 (5'pr3/3'pr1),
pCCAT237 (5'pr4/3'pr1), pCCAT190 (5'pr5/3'pr1), pCCAT469*
(5'pr1/3'pr2), and pCCAT434* (5'pr1/3'pr3). To create pCCAT162 the
naturally occurring StuI (-GGA/CCT-) restriction site at
position 55 of pCCAT493 was used. To introduce the
14 mutation in
pCCAT493, two fragments of the human protein C promoter region,
spanning nucleotides
396 to
33 (fragment 1) and nucleotides
13 to
+122 (fragment 2), were amplified with primers 5'pr6/3'pr4 and
5'pr7/3'pr5, respectively. Both PCR fragments 1 and 2 were digested by
Eco1CRI and equal amounts were ligated for 16 h at room
temperature. The ligation mixture was PCR-amplified with primers 1 and
3 and the 472-base pair fragment, consisting of protein C promoter
regions
396 to
42 and
5 to +107 separated by an
Eco1CRI site, was digested by SmaI. Subsequently,
the 457-base pair fragment was cloned in the SmaI site of
the CAT00 vector. This reporter construct was named pC
-41/-5CAT493.
The double-stranded oligonucleotide
5'-GGCCAAGCAAATATTTGTGGTTATGGACTAACTCGAA-3' (-41 to -5) was
cloned into the Eco1CRI site of pC
41/
5CAT493. The
integrity of all constructs was verified by sequencing.
Transient Transfection--
The human hepatoma cell line HepG2
(ATCC no. HB8065) and the SV40-transformed African green monkey kidney
cell line COS 7 (ATCC no. 1651-CRL) were cultured in minimal essential
medium containing Earle's salts and nonessential amino acids
supplemented with 15% heat-inactivated fetal calf serum. Cells were
seeded at a density of approximately 1 × 105
cells/60-mm tissue culture dish. After 24 h, a DNA mixture
containing 6 µg of protein C-CAT reporter construct, 2 µg of
-galactosidase expression vector (pCH110) (20), and 1.5 µg of
nonspecific plasmid pUC13, was transfected into the cells by the
calcium phosphate co-precipitation method (21). For cotransfection
experiments, 1 µg of HNF-1
expression vector and/or 2 µg of
HNF-6 expression vector were added, unless stated otherwise.
Forty-eight hours after transfection, cells were harvested, and
-galactosidase activity was measured (22). The CAT activity of each
construct was determined as described previously (23) and normalized to
-galactosidase activity. All transfections were repeated two to six
times in duplicate, with at least two different plasmid preparations,
and data from representative experiments are shown.
In Vitro Transcription/Translation-- HNF-6 was in vitro transcribed and translated using the TnT SP6 coupled wheat germ system (Promega Corporation BNL, The Netherlands) according to the manufacturer's protocol, in a final volume of 50 µl (17). The crude wheat germ extract containing translated protein was used directly in electrophoretic mobility shift assays (EMSAs).
Preparation of Cell Extracts-- Rat liver nuclear extracts were prepared as described (24). HNF-1- or HNF-6-containing COS 7 cell extracts were prepared as follows. The cells (6 × 105) were transfected for 6 h in Dulbecco's modified Eagle's medium without fetal calf serum by lipofection using DOTAP and 10 µg of pRSV-HNF-1 or pECE-HNF-6. Forty-eight hours after transfection, the COS 7 cells were washed with phosphate-buffered saline and harvested in 1 ml of 40 mM Tris-Cl (pH 7.5), 1 mM EDTA, 150 mM NaCl. The cells were pelleted and resuspended in 200 µl of 50 mM Tris-Cl (pH 7.9), 500 mM KCl, 0.5 mM EDTA, 2.5 µg/ml leupeptin, 1 mM dithiothreitol, 0.1% (v/v) Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and 20% (v/v) glycerol. After three freeze-thaw cycles, the lysates were centrifuged and the supernatants were collected. Human hepatoma HepG2 cell extracts were prepared following the same procedure as for COS 7 cell extracts.
Oligonucleotides Used in EMSAs--
Human protein C gene: pCwt
5'-TTTGTGGTTATGGATTAACTCGAACT-3' (28 to
3); pCmt
5'-TTTGTGGTTATGGACTAACTCGAACT-3' (
28 to
3); rat
pyruvate kinase gene: PK-H1, 5'-CTAGCTGGTTATACTTTAACCAGG-3' (
96 to
73); rat HNF-3
gene: HNF-3
,
5'-AGCTTAAGGCCCGATATTGATTTTTTTTTCTCC-3' (
150 to
118); rat
2-urinary globulin gene: UG-HNF-6, 5'-AAATGTATTATTGATAAAATCAAT-3' (
202 to
179). The
14 T
C mutation is underlined in the pCmt oligonucleotide.
EMSA--
EMSAs were performed with 3 µl of liver nuclear
extract, 3 µl of in vitro transcribed/translated HNF-6,
and 5 µl of COS 7 or HepG2 cell extract, in a 20-µl reaction
mixture containing 20 mM HEPES (pH 7.6), 2 mM
MgCl2, 0.5 mM EGTA, 1 mM
dithiothreitol, 10% (v/v) glycerol, and 1 ng of
32P-end-labeled oligonucleotide. After an incubation of 20 min on ice, free DNA and DNA-protein complexes were separated by
electrophoresis on a 6 or 8% polyacrylamide gel with 0.5× TBE buffer
at 4 °C. Competing oligonucleotides (50-fold molar excess) or
preimmune or immune serum were added to the incubation mixture 20 min
prior to addition of the 32P-labeled probe. After
electrophoresis, the gel was dried and subjected to autoradiography at
80 °C for 16 h.
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RESULTS |
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As shown in Fig. 1, the sequence of the HNF-1 site identified previously (13) in the human protein C gene promoter is compatible with the consensus reported for HNF-6 binding (17, 25). To determine whether HNF-6 actually binds to this region of the protein C promoter, EMSAs were performed with recombinant HNF-6. As shown in Fig. 2A (lane 2), HNF-6 transcribed/translated in a wheat germ extract formed a complex with the pCwt oligonucleotide. This complex was absent when unprogrammed wheat germ extracts were used as a source of proteins (Fig. 2A, lane 1). To confirm that this complex was due to HNF-6 binding, the experiments were repeated with a labeled probe (UG-HNF-6) known to bind HNF-6 (25). As shown in Fig. 2A (lanes 5-7), the complex formed between HNF-6 and the UG-HNF-6 oligonucleotide was competed not only with the corresponding unlabeled oligonucleotide, but also with the unlabeled pCwt oligonucleotide.
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Next, the pCwt oligonucleotide was tested in EMSAs with liver nuclear
extracts as a source of proteins. Several complexes were detected (Fig.
2B, lane 1). One complex (labeled
HNF-1) was prevented by an excess of the unlabeled
HNF-1-binding oligonucleotide PK-H1 (26) (Fig. 2B,
lane 2). Also, this complex co-migrated with that formed
between the probe and recombinant HNF-1 produced in COS 7 cells
transfected with an HNF-1 expression vector (Fig. 2B,
lanes 1, 6, and 8). Consistent with
previous results (13), it was concluded that this complex, seen with
liver extracts, results from HNF-1 binding to the probe. Another
complex (labeled HNF-6) was prevented by the addition of a
competing amount of oligonucleotide HNF-3, previously shown to bind
HNF-6 (25) (Fig. 2B, lane 3). This complex was
inhibited by addition of anti-HNF-6 serum in the binding reaction, but
not by addition of preimmune serum (Fig. 2B, lanes
4 and 5). In addition, the complex formed between the
probe and recombinant HNF-6 obtained by transfection of COS 7 cells,
co-migrated with the complex seen with liver nuclear extracts (Fig.
2B, lanes 1, 6, and 7). The
conclusion from these observations was that the complex seen with liver
nuclear extracts results from binding of HNF-6 to the pCwt probe. Two
minor complexes (Fig. 2B, open arrowheads) were
also observed. These complexes are specific as an excess of unlabeled
probe (data not shown) prevented them. The identity of the proteins
involved was not investigated in the present work. Finally, incubation
of the pCwt probe with HepG2 cell extracts produced two complexes that
co-migrate with those obtained with recombinant HNF-1 and HNF-6,
suggesting that the latter proteins are also expressed in this human
hepatoma cell line (Fig. 2B, lane 9).
The question as to whether HNF-1 and HNF-6 binding to the probe is
mutually exclusive was then investigated. EMSAs were performed with
binding reactions containing a fixed amount of HNF-6-containing COS 7 cell extracts and increasing amounts of HNF-1-containing extracts. As
shown in Fig. 3 (lanes 1-6),
increasing amounts of HNF-1 inhibited formation of the HNF-6-probe
complex, without production of a ternary complex. The reverse
experiment was also performed. Addition of increasing amounts of
HNF-6-containing COS 7 cell extracts to a mixture containing a fixed
amount of HNF-1-containing extracts led to inhibition of HNF-1-probe
complex formation (Fig. 3, lanes 7-12). These experiments
showed that binding of HNF-6 and HNF-1 to the 22 to
10 region of
the protein C gene promoter is mutually exclusive.
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To determine the influence of the 14 T
C promoter mutation on the
binding affinity of HNF-6 for the protein C promoter, EMSAs were
performed with an oligonucleotide containing an identical mutation
(pCmt). This mutation clearly reduced HNF-6 binding (Fig. 2A, lane 4). Moreover, HNF-6 binding to the
UG-HNF-6 oligonucleotide could be totally prevented by the
corresponding unlabeled oligonucleotide and by the unlabeled pCwt
oligonucleotide, but not by the pCmt oligonucleotide (Fig.
2A, lanes 5-8).
The next question was whether HNF-6 was able to transactivate the
protein C promoter. HepG2 and COS 7 cells were transfected with the
protein C reporter construct pCCAT493 and an expression vector for
HNF-6. As shown in Fig. 4A,
transfection of HepG2 cells with HNF-6 increased transcription from the
wild-type protein C promoter approximately 3-fold. When a construct
containing the 14 T
C mutation was used, basal activity was
severely reduced and transactivation by HNF-6 was abolished. Similarly,
in COS 7 cells the wild-type, but not the mutant promoter, was also
stimulated by HNF-6 (Fig. 4B). As expected, HNF-1 also
transactivated the reporter construct in COS 7 cells. However,
overexpression of HNF-1 in HepG2 cells resulted in a significant
decrease in protein C promoter activity. Dose-response experiments
(Fig. 5) showed that this inhibitory
effect started with as little as 0.5 µg of HNF-1 expression vector.
These observations contrasted with those of Berg et al.
(13), who showed that the protein C gene promoter is transactivated
1.5-fold by HNF-1 in HepG2 cells. This discrepancy could have been due
to the absence of the nontranslated first exon (nucleotides +1 to +52)
or the first part of intron 1 in the protein C reporter construct used
by Berg et al. (13), which ranges from nucleotides
618 to
+7. The experiments were therefore repeated in HepG2 cells with
constructs partially devoid of exon/intron 1 (pCCAT469* and pCCAT434*).
As shown in Fig. 6, both deletion constructs displayed an approximately 2-fold lower protein C promoter activity compared with the wild-type promoter. The effect of HNF-1 on
these 3' deletion constructs was then determined. In contrast to the
exon/intron 1-containing pCCAT493 construct, both pCCAT469* and
pCCAT434* were transactivated about 3-fold by the addition of HNF-1
(Fig. 6).
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The observation that HNF-1 activity was controlled by neighboring
3'-cis sequences led to a search for other modulatory sequences. A
number of 5'-deletion constructs were tested for both basal activity
and transactivation by HNF-1. As shown in Fig. 6, deletion of the 386
to
185 region (pCCAT291) slightly reduced basal transcriptional activity, while deletion of the
184 to
161 region (pCCAT267) had no
effect on basal activity. Cotransfection experiments with HNF-1
resulted for both pCCAT291 and pCCAT267 in a clear reduction in
promoter activity. Further deletion of nucleotides
160 to
131
(pCCAT237) slightly reduced basal activity. However, cotransfection with the HNF-1 expression vector up-regulated the activity of pCCAT237.
Deletion of nucleotides
130 to
84 (pCCAT190) had no effect on basal
activity, whereas the deletion of nucleotides
83 to
56 (pCCAT162)
resulted in an approximately 4-fold reduction in protein C promoter
activity. The influence of the
83 to
56 region was consistent with
data from Miao et al. (14). The expression of both pCCAT190
and pCCAT162 was up-regulated by addition of the HNF-1 expression
vector.
Finally, experiments were conducted to determine whether the sequences involved in the control of HNF-1 activity also influenced the HNF-6 response. HepG2 cells were cotransfected with each protein C reporter construct and an expression vector for HNF-6. This revealed that all constructs were transactivated by HNF-6. However, the full length construct (pCCAT493) and the two longest 5'-deletion constructs (pCCAT291 and pCCAT267) were transactivated to a slightly higher extent than the other deletion constructs (Fig. 6).
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DISCUSSION |
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The 14 T
C mutation in the protein C promoter region is
associated with type I protein C deficiency (13, 27). The present study
shows that the protein C promoter region contains an HNF-6 responsive
element at position
21 to
12. The
14 T
C mutation reduced the
binding of HNF-6 and abolished the transcriptional response to
overexpressed HNF-6. The HNF-6 responsive element completely overlaps
with a previously described HNF-1 responsive element, located at
position
22 to
10 (13). It was shown earlier that the
14 T
C
mutation abolishes binding and activity of HNF-1 (13).
The EMSAs reported here did not show formation of a ternary complex
between HNF-6, HNF-1, and the DNA probe. This strongly suggests that
HNF-6 and HNF-1 bind in a mutually exclusive way to the protein C gene
promoter. Apparently both transcription factors compete for binding to
the protein C promoter. The results also suggest that sequences located
upstream and downstream of the HNF-6/HNF-1 site increase the basal
activity of the promoter and inhibit the effect of HNF-1. Indeed,
deletion of a 24-base pair intronic region from the 3' part of the
reporter construct (pCCAT469*) or deletion of a 30-base pair region
(nucleotides 160 to
131) from the 5' part of the construct
(pCCAT237) decreased promoter activity and converted the inhibitory
effect of HNF-1 to a stimulatory effect. Examination of the intronic
region reveals the presence of a -GGGTGTGG- sequence at position
nucleotides +83 to +90, which resembles a TEF-2 consensus sequence
(28). The nucletotides
160 to
131 region contains two out of three sequence polymorphisms (C/T at nucleotides
153 and A/G at nucleotides
140) (29), which have been shown to be associated with variations in
protein C plasma levels and thrombotic risk (30). Furthermore, this
region contains a 5'-GGCAGAGGT-3' sequence, located at position
139
to
131, which is conserved between
species.2 The role of the
putative intronic TEF-2 site and of the upstream polymorphic and
conserved sequences in protein C promoter regulation requires further
investigation.
At present, there is no good explanation for the difference in HNF-1 effects observed with different promoter constructs. One possibility is that in transfected cells HNF-6 displaces HNF-1 from the protein C promoter and acts as a stronger transactivator. In this case, displacement may depend on the length of the promoter in the construct. This would be in line with the observations by Tsay et al. (16) who showed that binding of transcription factors to the protein C promoter involves synergistic interactions. Alternatively, factors that bind to the longer promoter constructs, which tend to possess stronger basal activity, may specifically be squelched by overexpressed HNF-1. Whatever the explanation, the question remains as to whether HNF-1, HNF-6 or both are important for protein C promoter activity in vivo. Experiments aimed at identifying the occupancy of the protein C promoter in intact tissues, based on in vivo footprinting, might resolve this intriguing issue.
When introduced in a promoter context that cannot be activated by
HNF-1, the 14 T
C mutation still induced a severe drop in basal
activity. This mutation also led to the loss of HNF-6 binding to, and
activation of, the protein C promoter. The activity of HNF-6 was
clearly less context-dependent than that of HNF-1. This
study therefore questions the role of HNF-1 in protein C promoter
activity and uncovers a function for HNF-6. In addition, this is the
first report describing the putative involvement of HNF-6 and of an
HNF-6 binding site in human pathology.
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ACKNOWLEDGEMENTS |
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We thank L. Nolte for HNF-1-containing COS 7 cell extracts and M. Pontoglio and M. Yaniv for pRSV-HNF1.
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FOOTNOTES |
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* This work was supported in part by Grant 92.004 from the Trombosestichting Nederland and by grants from the Belgian State Program on Interuniversity Poles of Attraction, from the D. G. Higher Education and Scientific Research of the French Community of Belgium, from the Fund for Scientific Medical Research (Belgium), from the National Fund for Scientific Research (Belgium), and from the Fonds de Développement Scientifique (Louvain University).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These two authors equally contributed to this work.
¶ To whom correspondence should be addressed: Dept. of Internal Medicine, Academic Medical Center, Rm. G1-113, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Tel: 31-20-5663224, Fax: 31-20-6977192, E-mail: C.A.SPEK@AMC.UVA.NL
** Recipient of a fellowship from the Fonds pour la Formation à la Recherche dans l'Industrie et l'Agriculture (Belgium).
Research Associate of the National Fund for Scientific Research
(Belgium).
1 The abbreviations used are: HNF, hepatocyte nuclear factor; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay.
2 C. A. Spek, R. M. Bertina, and P. H. Reitsma, manuscript submitted for publication.
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REFERENCES |
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