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Blood, Vol. 95 No. 6 (March 15), 2000:
pp. 2008-2014
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
The anticoagulant factor, protein S, is produced by cultured human
vascular smooth muscle cells and its expression is up-regulated by
thrombin
Omar Benzakour and
Chryso Kanthou
From the Molecular Cell Biology Laboratory, Thrombosis Research
Institute, London, UK.
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Abstract |
The anticoagulant factor protein S is a secreted vitamin K-dependent
 -carboxylated protein that is mainly made in the liver. Protein S is
homologous to the growth arrest specific protein, Gas6, the expression
of which is up-regulated in cultured fibroblasts upon serum withdrawal.
We report here the synthesis and secretion of protein S by cultured
human vascular smooth muscle cells (HVSMCs). Western blot analysis
revealed that similar amounts of protein S are secreted by both
growing and growth-arrested HVSMCs. HVSMC-derived protein S was
found to be -carboxylated as it was precipitated by barium citrate
and was shown to possess protein C cofactor activity. Treatment with
the vitamin K antagonist warfarin led to the accumulation of
intracellular undercarboxylated protein S forms that were rapidly
secreted upon the reintroduction of vitamin K. Northern blotting
analysis showed that cultured HVSMCs express a protein S
transcript. The expression of protein S messenger RNA was
unaffected by either warfarin, growth arrest, or various VSMC mitogens,
such as platelet-derived growth factor-BB, basic fibroblast growth
factor, transforming growth factor- , or hepatocyte growth factor.
Thrombin, however, induced an up-regulation of protein S expression at
both messenger RNA and protein levels. The evidence we provide for
protein S secretion by cultured HVSMCs and its up-regulation by
thrombin, together with earlier reports showing that protein S acts as
a mitogen for these cells, suggests that, in addition to its known role
in regulating blood clotting, protein S may also be an important
autocrine factor in the pathophysiology of the vasculature.
(Blood. 2000;95:2008-2014)
© 2000 by The American Society of Hematology.
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Introduction |
Protein S is a 69 kd single-chain plasma
glycoprotein that acts as a cofactor for activated protein C in the
inactivation of coagulation factors Va and VIIIa.1 It
requires no activation by proteolytic cleavage and circulates at a
concentration of about 270 nmol/L in both a free active form (40%) and
in an inactive form (60%) bound to C4b-binding protein.2
Protein S belongs to the family of mainly secreted vitamin K-dependent
proteins that includes a number of zymogens and cofactors of the
coagulation cascade in which some glutamyl residues are
posttranslationally modified to -carboxyglutamic acid (Gla)
residues.3 The Gla posttranslational modification is
catalyzed by a microsomal -glutamyl carboxylase that requires
vitamin K in its reduced form as a cofactor.4 The enzymatic
reaction generates -carboxyglutamate and vitamin K 2,3,-epoxide that
is then recycled back to the hydroquinone form by a reductase
enzyme.4 Warfarin
(3-[ -acetonyl-benzyl-4-hydroxycoumarin]), a vitamin K
antagonist, binds and inhibits the activity of the vitamin K epoxide
reductase and thus blocks the vitamin K cycle.3,4 This
property of warfarin has led to its use in anticoagulant therapy.5 Gla residues allow Ca++-dependent
protein-phospholipid complex formation and facilitate conformational
changes that are essential for zymogen activation or for cofactor
activity.6 Following the Gla region is a domain unique to
protein S, which consists of a disulphide loop that is highly
susceptible to proteolysis by serine proteinases such as
thrombin.7-9 This domain is followed by four
epidermal growth factor-like domains. The C-terminus of protein S is
unrelated to other coagulation factors and is homologous to the plasma
sex hormone binding globulin8 and the basement membrane
proteins laminin A and merosin.10 The latter two proteins
have been shown to play a role in cell proliferation, migration, and
differentiation.10
The major producer of protein S and other factors of the coagulation
system is the liver.3 However, protein S is also
synthesized at extrahepatic sites, including the brain and
spleen11,12 as well as endothelial cells,13,14
megakaryocytes,15 and osteoblasts,16 and cells
of the nervous system,17 suggesting that it has functions
distinct from its anticoagulant activities. The product of a gene
specifically expressed during serum starvation in fibroblasts, named
growth arrest specific gene 6 (Gas6), represents a new member of the
vitamin K-dependent protein family that is homologous to protein
S.18 Apart from its lack of a thrombin-cleavage site, Gas6
protein exhibits strong homology with all the other structural domains
of protein S.18 Gas6 is synthesized and secreted by
cultured vascular smooth muscle cells (VSMCs) and was shown to
potentiate thrombin-induced proliferation in these
cells.19,20 These effects of Gas6 are dependent on its
-carboxylation as Gla-deficient Gas6 lacks receptor-binding and
growth potentiating activities.20,21 Gas6 was also shown to
prevent fibroblast, endothelial, and VSMC death by apoptosis and hence
was postulated to be a cell survival factor.22-24
Gasic et al25 and our laboratory26 have
previously demonstrated that protein S induces the proliferation of
cultured rat and human VSMCs (HVSMCs), respectively, and evidence for
the existence of a specific protein S receptor(s) on HVSMCs has been
provided.26 The cellular activities of protein S on VSMCs
in vitro occur within its circulating range2 (270 nmol/L),
thus suggesting that such activities may occur in vivo. In the present
report, we show that cultured HVSMCs produce and secrete protein S into
their conditioned media and express a protein S transcript. The
expression of protein S by serum-starved, growth-arrested, and growing
VSMCs as well as the regulation of its synthesis and secretion by
vitamin K, warfarin, and various cytokines was investigated.
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Materials and methods |
Materials
Purified human -thrombin (3000 U/mg) and human protein S were
from Enzyme Research Laboratories (UK). Recombinant human
platelet-derived growth factor-BB (PDGF-BB) and basic fibroblast growth
factor (b-FGF) were from Bachem (UK). Human recombinant hepatocyte
growth factor, human platelet transforming growth factor beta-1,
vitamin K1, and 3-(-acetonylbenzyl)-4-hydroxy-coumarin (warfarin)
were from Sigma (UK). Tritiated thymidine ([3H]-TdR; 120 Ci/mmol/L), [-32P]-dCTP (>3000 Ci/mmol/L), and
[35S]-methionine/[35S]-cysteine
(Pro-MixTM) were from Amersham-Pharmacia Biotech (UK). Rabbit
antihuman protein S antibody and horseradish peroxidase-conjugated goat
antirabbit or antimouse immunoglobulin G were from Dako (UK). Monoclonal antibodies to protein S Gla domain (HSP 21) and epidermal growth factor-like region (HSP 41) were a kind gift from Professor Bjorn Dahlbäck (Department of Clinical Chemistry, University of
Lund, Malmö, Sweden).8
Cell culture
HVSMCs isolated from normal abdominal artery were grown as
monolayers in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), subcultured at a split ratio of 1:4,
and used between passages 2-8 as described previously.26,27
Analysis of protein S synthesis and secretion
Protein S production was assessed in conditioned media and cell
lysates. Two cell culture conditions were used: growth-arrested and
growing HVSMCs. Growth-arrested cells were obtained by transferring confluent cultures (grown at about 90% confluency in their
characteristic "hills and valleys" pattern)26,27 into
serum-free DMEM for 48 hours prior to the commencement of media
conditioning. Growing cells were subconfluent cultures (around 60%
confluency) initially grown in serum that were placed in a defined
serum-free growth medium (to avoid possible interference because of
serum-derived protein S) at the start of media conditioning. The
serum-free growth promoting medium consisted of DMEM containing a
supplement of insulin (5 µg/mL), transferrin (5 µg/mL), sodium
selenite (5 ng/mL) (Sigma, UK), PDGF-BB (2 ng/mL), and b-FGF
(2 ng/mL). These conditions were found to support the growth and
proliferation of HVSMCs in the absence of any serum supplement for 3 to
5 days. To confirm the growth state of the cells, parallel cultures
were set up and used to monitor levels of [3H]-TdR
incorporation. Cells were pulse-labeled for 4 hours with 5 µCi/mL [3H]-TdR at 24-hour intervals, and
[3H]-TdR incorporation was measured as described
previously.26-28 Under these conditions, the levels of
[3H]-TdR incorporation for growing cells expressed as
average counts per minute from 3 determinations were as follows:
13 460 ± 945 at day 1; 15 890 ± 950 at day 2; and
20 250 ± 765 at day 3. For growth-arrested cells, levels of
[3H]-TdR incorporation in counts per minute determined at
daily intervals after the 48-hour growth-arrest period were:
3765 ± 408 at day 1; 1659 ± 98 at day 2; and
1275 ± 108 at day 3. Fresh serum-free DMEM (or DMEM with growth
supplements described above for growing cells) was added to the cells
at a ratio of 5 mL/106 cells for specified periods of time
after which conditioned media were collected. The influence of vitamin
K1 (1-10 µg/mL), of warfarin (1 µg/mL), or of specific growth
factors or cytokines on protein S production was determined by
including these agents in serum-free media. Proteins in conditioned
media and cell lysates were quantified using a Biorad microassay. To
take into account differences in cell numbers between the various cell
culture conditions used (growth-arrested vs growing cells or cells
treated with various growth factors), for each individual experiment
and/or time point reported, the volumes of conditioned media that were
analyzed were adjusted to total cellular protein.
Western blotting analysis of protein S synthesis and secretion
Proteins in conditioned media were precipitated with trichloroacetic
acid, washed in acetone, and resuspended in Laemmli sample buffer.29 Cell monolayers were lysed directly in sample
buffer. Protein S expression was analyzed by Western blotting, using
polyclonal and monoclonal antiprotein S antibodies as detailed
previously.8 Proteins were visualized by enhanced
chemiluminesence ECL (Amersham Pharmacia Biotech). To exclude the
possibility that the protein S detected was a contaminant from serum,
metabolic labeling and immunoprecipitation experiments using
antiprotein S antibodies followed by SDS-PAGE and autoradiography were
performed as described by Wu et al.30
Determination of the extent of -carboxylation of secreted protein
S
The selective precipitation of Gla proteins in conditioned media by
barium citrate was performed as described by Berkner.31 Conditioned media were treated with sodium citrate (20 mmol/L) and
BaCl2 (40 mmol/L) and incubated on ice for 1 hour. The
precipitate was collected by centrifugation, washed in 100 mmol/L
BaCl2/100 mmol/L NaCl, and resolubilized in
phosphate-buffered saline containing 150 mmol/L sodium citrate and
0.1% bovine serum albumin. Supernatants and resolubilized precipitates
were further analyzed by Western blotting as described
previously.8
Northern blotting
Total RNA was extracted using an Rneasy RNA extraction kit (Qiagen,
UK). Hybridization was performed using a human protein S complementary
DNA probe, kindly provided by Professor Bono Bouma (University of
Utrecht, The Netherlands) that was labeled with [-32P]-dCTP as we described previously.27
Membranes were stripped and reprobed with a G6PD complementary DNA
probe to normalize for equal loading, and autoradiograms were
densitometrically scanned.
Quantification and cofactor activity assays
Secreted protein S was quantified using an
enzyme-linked immunosorbent assay (ELISA) kit (Diagnostica-Stago,
France) according to the manufacturer's instructions, except that
purified protein S was used as a calibrator in place of the supplied
serum standard. Conditioned media were treated with a cocktail of
protease inhibitors (CompleteTM, EDTA-free, Roche Diagnostics, UK)
and concentrated 10-fold using 3000 Da molecular weight cut-off
Microcon 3 microconcentrators (Amicon, USA) prior to being assessed for
their protein S content. The functional activity of cell-secreted
protein S was determined using a "Statclot" clotting
assay kit (Diagnostica-Stago) according to the manufacturer's
instructions, except that purified protein S was used for the
construction of the calibration curve. Concentrated conditioned media
were buffer-exchanged in phosphate-buffered saline, and aliquots were
assessed for their cofactor activity expressed as a prolongation in
clotting time. Average values from four determinations expressed as
prolongation in clotting times obtained with ng quantities of purified
protein S in the calibration curve were as follows: 25 ng, 12 ± 4
seconds; 50 ng, 22 ± 3 seconds; 100 ng, 37 ± 4 seconds; and
200 ng, 53 ± 2 seconds).
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Results |
Synthesis and secretion of protein S by cultured HVSMCs
The presence of protein S in cultured HVSMC-conditioned media was
first analyzed by Western blotting. Figure
1 shows that both growth-arrested and
growing cells secrete protein S that is detected as a single band of 70 kd. This 70-kd protein was specifically
recognized by both anti-protein S monoclonal (HSP41 and
HSP21)8 and a rabbit polyclonal antibody. The results shown in Figure 1 were obtained with the polyclonal antibody that was used
subsequently in our study. The above antibodies did not detect purified
human Gas6 that is also known to be produced by VSMCs in culture. The
possibility that protein S detected in HVSMC-conditioned media could be
a serum contaminant was ruled out. Metabolic labeling experiments were
performed in which HVSMCs were labeled with a mixture of
[35S]-methionine and [35S]-cysteine for 24 hours after which conditioned media were collected and subjected to
immunoprecipitation followed by SDS-PAGE and autoradiography (data not
shown). These experiments confirmed the data obtained by Western
blotting, as similar amounts of a single 70-kd
[35S]-labeled protein were detected in both growing and
growth-arrested HVSMC conditioned media.
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| Fig 1.
Protein S production by growing and growth-arrested
human vascular smooth muscle cells (HVSMCs).
Protein S production was assessed in conditioned media. Two cell
culture conditions were used: growth-arrested (GA) and growing (GR)
cells. Growth-arrested cells were obtained by transferring 90%
confluent cultures into serum-free Dulbecco's modified Eagle's medium
(DMEM) for 48 hours prior to the commencement of media conditioning.
Growing cells, were subconfluent cultures (60% confluency) that were
placed in a defined growth promoting serum-free medium at the start of
media conditioning. Conditioned media were collected after 24 hours
(lanes 1, 4), 48 hours (lanes 2, 5), and 72 hours (lanes 3, 6) and were
analyzed by Western blotting using a rabbit anti-protein S antibody.
Purified 1 ng protein S (pS) and 50 ng recombinant human growth arrest
specific protein 6 (G6) were included as controls.
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The quantities of protein S made by HVSMCs in culture were estimated by
both ELISA and Western blotting. From such experiments, it was
estimated that HVSMC cultures secreted on average 40 ± 0.3 ng of
protein S/106 cells/24 h. The functional cofactor activity
of HVSMC-secreted protein S was investigated using the
"Statclot" protein S clotting assay and 10-fold
concentrated HVSMC-conditioned media obtained after a 72-hour
incubation in serum-free DMEM. A prolongation of 18 ± 3 seconds
in clotting activity was obtained with three separate concentrated
conditioned media samples each containing 50 ng of HVSMC-derived
protein S as estimated by ELISA. As a comparison, in this assay, the
prolongation in clotting observed with 50 ng purified human
protein S was 22 ± 3 seconds (n = 4). Because the -carboxylation of protein S is required for its cofactor
activity,1 it may be deduced that HVSMC-secreted
protein S had been fully processed.
Relationship between protein S synthesis and secretion and its
-carboxylation state
Growth-arrested HVSMCs in serum-free medium were treated with either
vitamin K (1 µg/mL), warfarin (1 µg/mL), or diluent for 48 hours
after which the presence of protein S in conditioned media and cell
lysates was analyzed by Western blotting. Figure 2A shows that warfarin treatment resulted
in a drastic decrease in the levels of protein S detected in
HVSMC-conditioned media that was paralleled by its intracellular
accumulation, hence implying that warfarin prevents protein S
secretion. It should be noted here that several cell types in culture
take up vitamin K from serum and have the capacity to recycle and reuse
it.31,32 Nevertheless, as an additional control, we have
added exogenous vitamin K to cells kept in serum-free medium. As
expected, the addition of extra exogenous vitamin K had no significant
effect on the secretion or intracellular expression of protein S as
compared with control cells.
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| Fig 2.
Effect of warfarin on protein S secretion.
A. Human vascular smooth muscle cell (HVSMC) cultures at 90%
confluency were incubated for 72 hours in control serum-free medium (C)
or medium containing warfarin (1 µg/mL) (W) or vitamin K (1 µg/mL)
(K). Conditioned media (CM) and cell lysates (CL) were analyzed by
Western blotting using a rabbit anti-protein S antibody. B. HVSMC
cultures at 90% confluency were incubated first for 72 hours with
either control medium (C) or medium containing warfarin (1 µg/mL)
(W); cells were then placed in new control medium (C) or medium
containing vitamin K (10 µg/mL) (K), which was collected after 4 hours and analyzed by Western blotting using a rabbit anti-protein S
antibody.
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The inhibition of protein S secretion by warfarin is a reversible
process. Indeed, Figure 2B shows that, if cells that had been treated
with warfarin for 72 hours were switched to a medium containing a
10-fold excess of vitamin K, intracellularly accumulated protein S was
released within 4 hours. No detectable protein S was found in
conditioned media collected over a 4-hour period from either control
cells or cells previously treated with warfarin and then placed in
control medium, thus excluding the possibility that the protein S
detected on the reintroduction of vitamin K was due to new synthesis
and secretion. The possibility that the observed inhibition of protein
S secretion by warfarin was due to an overall nonspecific inhibition of
total protein secretion was ruled out. Indeed, HVSMC cultures were
metabolically labeled with [35S]-methionine and
[35S]-cysteine in the presence of either vitamin K,
warfarin, or control for 48 hours. No differences in the total protein
secretion were observed between control, vitamin K-, or
warfarin-treated cells (data not shown). Taken together, data presented
in Figures 2A and 2B demonstrate that undercarboxylated protein S
accumulates intracellularly in HVSMCs, implying that its
-carboxylation is a prerequisite for its secretion.
Further experiments were performed to study the secretion of
protein S and its inhibition by warfarin in growth-arrested HVSMC over longer periods of time. Figure 3 shows
that HVSMCs continue to secrete protein S for up to at least 9 days
following their growth arrest in serum-free medium. As expected,
vitamin K addition did not significantly change the levels of
protein S secretion, confirming that vitamin K was recycled by the
cells and hence is not a rate-limiting factor for protein S
-carboxylation. The inhibition of protein S secretion by warfarin
was also consistently observed over this period.
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| Fig 3.
Continued production of protein S by human vascular
smooth muscle cells (HVSMCs) following extended periods of growth
arrest.
HVSMC cultures at 90% confluency were placed in either control
serum-free medium (C), medium containing vitamin K (1 µg/mL) (K), or
warfarin (1 µg/mL) (W). Conditioned media were replaced every 3 days
and were analyzed by Western blotting using a rabbit anti-protein S
polyclonal antibody. pS represents 1 ng purified protein S.
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Gamma-carboxylated proteins are selectively precipitated by barium
citrate.31 To study further the relation between protein S
secretion and its state of -carboxylation, barium citrate
precipitation of conditioned media experiments was undertaken. Figure
4 shows that in control and vitamin
K-treated cells most of the secreted protein S was recovered in the
barium citrate precipitate fraction, inferring that this protein S was
-carboxylated. In contrast, in warfarin-treated cells, the small
amount of secreted protein S was not precipitated by barium citrate,
confirming that, in the presence of warfarin, protein S remained
undercarboxylated. Accordingly, when barium citrate precipitation was
performed on cell lysates of warfarin-treated cells, protein S was
exclusively found in the nonprecipitable fraction (data not shown).
Altogether the data presented above strongly suggest that HVSMCs
synthesize and secrete fully -carboxylated and functional protein S.
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| Fig 4.
Production of -carboxylated protein S by human
vascular smooth muscle cells (HVSMCs).
Conditioned media collected from 90% confluent HVSMC cultures
maintained in either control (C), warfarin (1 µg/mL) (W), or vitamin
K (1 µg/mL) (K) for 72 hours were subjected to barium citrate
precipitation as described in "Materials and methods." Aliquots
from total unprecipitated (T), barium citrate precipitated (P), and
nonprecipitable supernatant fractions (S) were analyzed by Western
blotting using an anti-protein S polyclonal antibody. Protein S (1 ng)
(pS) was used as a control.
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Regulation of protein S expression
In Northern blotting experiments, using a protein S complementary
DNA probe, the expression of protein S transcript in HVSMCs was
analyzed, and a single transcript of around 4 kB was detected. In Table
1, the ratio of protein S to the
housekeeping gene G6PD messenger RNA (mRNA) is represented. HVSMC
cultures were either placed in 10% FCS containing medium (growing) or
0.5% FCS (growth arrested). Thymidine incorporation experiments
confirmed that under these conditions, whereas cells in the presence of
10% FCS continued to grow and undergo cycles of DNA synthesis, those
placed in 0.5% FCS become growth arrested (data not shown) that is in agreement with previous studies.27,28,33 The level of
expression of the protein S transcript did not show any
significant variation between growing as compared with growth-arrested
cells, confirming the observations made at the protein level seen in
Figure 1. Neither treatment of cells for 24 or 48 hours with warfarin
or vitamin K resulted in any significant changes in protein
S transcript levels. These data indicate that protein S mRNA
levels are unaltered by the growth state of the cells or their vitamin
K status.
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Table 1.
Effect of growth conditions, warfarin, and vitamin K on
protein S messenger RNA expression in human vascular smooth muscle
cells (HVSMCs)*
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The possible regulation of protein S expression by some VSMC mitogens
(at concentrations known to induce maximal biological activity in
HVSMCs)27,28,33-35 was investigated. Growth-arrested HVSMCs
in serum-free medium were treated with either PDGF-BB (10 ng/mL), b-FGF
(5 ng/mL), transforming growth factor beta-1 (5 ng/mL), hepatocyte
growth factor (20 ng/ml), or -thrombin (0.5-5 U/mL) for 24 or 48 hours. In agreement with previous studies under these conditions, cells
responded mitogenically to the factors above27,28,33-35
(data not shown). The strongest mitogenic effect was observed with
PDGF-BB that induced a 7.8-fold increase in the level of DNA synthesis
as estimated by [3H]-TdR incorporation. Treatment of
HVSMCs with either PDGF-BB, b-FGF, hepatocyte growth factor, or
transforming growth factor beta-1 led to no significant changes in the
level of protein S secreted, shown after a 48-hour incubation (Figure
5A). However, as depicted in Figure 5B, the
serine proteinase, -thrombin, which is also a potent HVSMC
mitogen,27 led to both a significant increase in the level
of protein S secretion and its cleavage that was more pronounced when 5 U/mL thrombin was used.
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| Fig 5.
Regulation of protein S synthesis by growth factors and
thrombin.
A. Conditioned media collected from 90% confluent human vascular
smooth muscle cell (HVSMC) cultures maintained in serum-free medium for
48 hours in the presence of either control (lane 1), 5 ng/mL basic
fibroblast growth factor (b-FGF) (lane 2), 2 ng/mL transforming growth
factor beta-1 (TGF-)1 (lane 3), 5 ng/mL hepatocyte growth factor
(HGF) (lane 4), or 5 ng/mL platelet-derived growth factor-BB (PDGF-BB)
(lane 5) were analyzed by Western blotting using an anti-protein S
polyclonal antibody. B. Conditioned media were collected from 90%
confluent cultures maintained in serum-free medium for 24 hours in the
presence of either control (lane 1), -thrombin at either 0.5 U/mL
(lane 2), 1 U/mL (lane 3), or 5 U/mL (lane 4). Conditioned media were
analyzed by Western blotting using an anti-protein S polyclonal
antibody. Arrows indicate cleaved forms of protein S.
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The cleavage of various forms of plasma-derived and recombinant protein
S by thrombin has been extensively studied previously.9,36 Three potential thrombin-sensitive sites have been identified within
the thrombin-sensitive region of human protein S at Arg49, Arg60, and Arg70. Cleavage at one or more of
these sites results in the formation of a lower molecular weight
protein that corresponds to the band indicated by the top arrow in
Figure 5B at about 68 kd. More extensive proteolytic
cleavage of protein S by higher concentrations of thrombin was
previously9 demonstrated to result in the formation of a
protein band of 54 kd that corresponds to the
protein indicated by the middle arrow in Figure 5B. A third band of
about 50 kd was also detected, indicated by the lower arrow in Figure 5B, that could have resulted from further proteolytic cleavage after exposure of other thrombin-sensitive sites or
alternatively via cleavage by other cell secreted thrombin-induced
proteases. Figure 6 represents Northern
blotting analysis of thrombin effects on protein S transcript
expression. A marked increase in protein S mRNA levels was evident at
12 hours, peaked at 24 hours, and then declined at 48 hours after
thrombin addition (5 U/mL) to growth-arrested HVSMCs. Therefore, it can
be concluded that the observed increase in the level of protein S
secretion by thrombin is accompanied by an up-regulation of
protein S gene expression and of mRNA stabilization.
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| Fig 6.
Northern blot analysis of regulation of protein S
messenger RNA expression by thrombin.
Human vascular smooth muscle cells (HVSMCs) at 90% confluency were
incubated with -thrombin (5 U/mL) for the indicated times. Total RNA
was electrophoresed on 1.2% agarose gel, transferred to a nylon
membrane, and sequentially hybridized with 32P-labeled
protein S and G6PD complementary DNA probes.
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Discussion |
In this report, it is demonstrated that cultured HVSMCs synthesize
and secrete the anticoagulant factor protein S. Warfarin inhibition of
the -carboxylation process attenuates protein S secretion resulting
in its intracellular accumulation. On the reestablishment of a normal
vitamin K status, this intracellular pool of protein S is rapidly
-carboxylated and secreted. Barium citrate precipitation experiments
together with the assessment of protein S cofactor activity in
conditioned media provided further confirmation that protein S secreted
by HVSMCs is effectively -carboxylated. In contrast to Gas6, the
expression of which was shown to be up-regulated by serum withdrawal in
fibroblasts,18 the secretion of protein S in HVSMCs was
similar in both growing and growth-arrested cells. Additionally, Nakano
et al20 have shown that Gas6 continues to be secreted by
rat VSMCs treated with warfarin. Therefore, the mechanisms that
regulate the secretion of Gas6 and protein S appear to differ.
Cellular, structural, as well as species differences have been shown to
mediate the secretory or degradation responses of Gla proteins to
warfarin.30,37 For example, in rat H-35 cells, warfarin
treatment causes an almost complete cessation of prothrombin secretion
with enhanced degradation of the intracellular pools,37
whereas in human HepG2 cells undercarboxylated prothrombin is secreted
in the presence of warfarin.30 When rat prothrombin was
transfected into warfarin-treated HepG2 cells, it was found to
accumulate intracellularly, indicating that the retention and
degradation of prothrombin by human hepatocytes is related to
structural determinants within the rat protein.30
The production of protein S by some fully differentiated
cells11-17 and as shown here by HVSMCs taken together with
evidence previously provided for its mitogenic
activity25,26 raise the possibility that locally produced
protein S may have local anticoagulant activity or other as yet unknown
functions. Several investigators22-24,38 have demonstrated
that Gas6 prevents cell death by apoptosis. Melaragno et
al39 showed in a rat model that both Gas6 and its tyrosine
kinase receptor Axl are strongly up-regulated in injured blood
vessels, suggesting an important role for this homologue of protein S
in vascular injury and repair. The sustained production of protein S by
cultured HVSMCs even several days after growth arrest may suggest a
role for this protein in cell survival. Protein S deficiency was
reported to be associated with osteopenia, osteonecrosis, and vascular
calcification.40-42 Locally produced protein S may also be
involved in vascular tissue calcification, evident in advanced
atherosclerotic lesions.43
The up-regulation of protein S expression by thrombin in HVSMCs may be
of crucial importance for both hemostasis and vascular repair
processes. Previous reports have indicated that thrombin is generated
during blood clotting at concentrations of up to 130 nmol/L, is
actively incorporated into clots, and is released during clot
retraction and fibrinolysis.44 Therefore, the concentration of thrombin (40 nmol/L) found here to result in optimal up-regulation of protein S expression would be expected to be present at sites of
vascular injury. Thrombin also causes protein S cleavage, thus rendering it inactive as an anticoagulant.9 Because
putative cellular functions for thrombin-cleaved protein S have not
been determined as yet, the significance of the observed protein S cleavage by thrombin remains to be unveiled. It will be of particular interest to investigate whether the up-regulation of protein S by
thrombin observed in VSMCs also occurs in hepatocytes that are the main
known producer cells of factors of the coagulation system, including
protein S.3 Scarpati and DiCorletto45 reported the presence of a thrombin responsive element within the PDGF-B gene
promoter that consists of a repeat of CCACCC in an ABBA configuration. Our computer search has revealed the existence of this putative thrombin responsive consensus sequence within the protein S gene promoter region. Functional studies of these putative thrombin responsive elements should lead to the identification of the
transcription factors specifically activated by thrombin in VSMCs and
that might mediate the observed up-regulation of protein S gene expression.
| |
Acknowledgments |
The authors are grateful to Professor Bjorn Dahlbäck, Professor
Bono Bouma, and Dr Brian Varnum for providing monoclonal antibodies to
protein S, a protein S complementary DNA probe, and human recombinant
Gas6 protein, respectively. The authors are also grateful to Professor
Fedor Bachmann and Dr Vincent Ellis for critical reading of the manuscript.
| |
Footnotes |
Submitted August 10, 1999; accepted November 29, 1999.
Supported by the British Heart Foundation PG95/138 and the Thrombosis
Research Trust. Salary for O.B. was supported in part by the Gary
Weston Foundation.
C.K.'s present address is Tumour Microcirculation Group, PO Box
100, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR, UK;
e-mail: kanthou{at}graylab.ac.uk.
Reprints: Chryso Kanthou, Tumour Microcirculation Group, PO Box
100, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR UK; e-mail:
kanthou{at}graylab.ac.uk.
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.
| |
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Abstract
Introduction