Reconstitution of the Human Endothelial Cell Protein C
Receptor with Thrombomodulin in Phosphatidylcholine Vesicles
Enhances Protein C Activation*
Jun
Xu
§,
Naomi L.
Esmon
¶, and
Charles T.
Esmon
§¶
**
From the
Cardiovascular Biology Research Program,
Oklahoma Medical Research Foundation, the Departments of
¶ Pathology and § Biochemistry and Molecular Biology,
University of Oklahoma Health Sciences Center, and the
Howard
Hughes Medical Institute, Oklahoma City, Oklahoma 73104
 |
ABSTRACT |
Blocking protein C binding to the endothelial
cell protein C receptor (EPCR) on the endothelium is known to reduce
protein C activation rates. Now we isolate human EPCR and
thrombomodulin (TM) and reconstitute them into phosphatidylcholine
vesicles. The EPCR increases protein C activation rates in a
concentration-dependent fashion that does not saturate at
14 EPCR molecules/TM. Without EPCR, the protein C concentration
dependence fits a single class of sites (Km = 2.17 ± 0.13 µM). With EPCR, two classes of sites
are apparent (Km = 20 ± 15 nM and
Km = 3.2 ± 1.7 µM). Increasing
the EPCR concentration at a constant TM concentration increases the
percentage of high affinity sites. Holding the TM:EPCR ratio constant
while decreasing the density of these proteins results in a decrease in
the EPCR enhancement of protein C activation, suggesting that there is
little affinity of the EPCR for TM. Negatively charged phospholipids
also enhance protein C activation. EPCR acceleration of protein C
activation is blocked by anti-EPCR antibodies, but not by annexin V,
whereas the reverse is true with negatively charged phospholipids.
Human umbilical cord endothelium expresses approximately 7 times more EPCR than TM. Anti-EPCR antibody reduces protein C activation rates
7-fold over these cells, whereas annexin V is ineffective, indicating
that EPCR rather than negatively charged phospholipid provide the
surface for protein C activation. EPCR expression varies dramatically
among vascular beds. The present results indicate that the EPCR
concentration will determine the effectiveness of the protein C
activation complex.
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INTRODUCTION |
The protein C anticoagulant pathway is critical to the negative
regulation of the blood clotting cascade (for review, see Refs. 1 and
2). The pathway is initiated when thrombin binds to thrombomodulin
(TM),1 an integral membrane protein on
endothelium and some other cell types.
This complex then converts protein C to activated protein C (APC),
which, in complex with protein S, inactivates factors Va and VIIIa
resulting in inhibition of thrombin generation.
Protein C is a vitamin K-dependent zymogen that, like other
vitamin K-dependent proteins, is capable of binding to
negatively charged phospholipid surfaces (3, 4). Unlike the other
vitamin K-dependent proteins, activation occurs on the
surface of endothelial cells. In vivo, endothelial cells
serve primarily anticoagulant functions. Because negatively charged
phospholipid would serve to augment the clotting cascade as well as the
anticoagulant pathway, for the endothelium to serve selective
anticoagulant functions alternative mechanisms are required for
assembling anticoagulant complexes on the endothelial cell surface.
Endothelial cells constitutively express a protein C/APC-binding
protein, designated the endothelial cell protein C receptor (EPCR) (5),
which appears to serve this function. Blocking protein C binding
to EPCR decreases protein C activation by the thrombin-TM complex on
cultured endothelium, primarily because of an increase in the
Km for protein C (6). In humans, the plasma
concentration of protein C,
65 nM, is considerably below
the Km for the thrombin-TM activation complex
incorporated into phosphatidylcholine vesicles (2 µM
(7)).
Because the vasculature varies dramatically in diameter between the
large vessels and the capillaries, assuming a constant TM density per
endothelial cell, the effective TM concentration rises from less than 5 nM to more than 100 nM as the blood moves from
the aorta to the capillaries (1, 8). This has led to the concept that
most protein C activation occurs in the microcirculation (1). These
considerations raise the question of whether there might be a
compensatory mechanism to augment protein C activation selectively in
the large vessels. EPCR expression varies throughout the vasculature
(9) and is sensitive to a variety of effector systems (5). In
vivo, EPCR expression levels are highest on the endothelium of
large vessels, decreasing progressively with decreasing vessel size
until the EPCR becomes undetectable by immunohistochemical approaches
in most capillary beds (9, 10). With cultured endothelium, EPCR
expression is down-regulated by cytokines like tumor necrosis factor
(5, 11). In contrast, EPCR mRNA levels are up-regulated by
thrombin in cell culture and by endotoxin and thrombin in
vivo (12). Because of the variable EPCR expression on endothelium,
the relationship between EPCR expression levels and its cofactor
function in protein C activation gains biological importance for
understanding the anticoagulant properties of different vascular beds.
To address the relationship between the EPCR concentration and protein
C activation, we have isolated EPCR from placenta and recombinant EPCR
from cultured cells and reconstituted the protein into phospholipid
vesicles. The results indicate that an excess of EPCR to TM is required
for optimal EPCR enhancement of protein C activation and that the EPCR
enhances activation effectively when reconstituted into
phosphatidylcholine vesicles that cannot effectively support the
coagulation cascade.
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EXPERIMENTAL PROCEDURES |
Materials--
Human protein C and APC (13), recombinant
Gla-domainless protein C (rGDPC) (14), bovine (15) and human (16)
thrombin, monoclonal antibodies (mAbs) against human EPCR (1494, 1495, 1496, 1500) (6), mAbs against human TM (CTM1009, CTM1029) (13), and
phospholipid vesicles (17) were prepared as described. Recombinant annexin V was a generous gift from Dr. Kazuo Fujikawa. The following reagents were purchased from the indicated vendors: Spectrozyme PCa,
American Diagnostica Inc.;
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine, and
1,2-dilinoleoyl-sn-glycero-3-phosphatidylethanolamine, Avanti Polar Lipids, Inc.; [14C]phosphatidylcholine, NEN
Life Science Products; iodine-125, Amersham Pharmacia Biotech;
octyl-
-D-glucopyranoside (OG), Calbiochem; Lubrol PX,
Triton X-100, chondroitinase ABC, Sigma; Affi-Gel 10, Bio-Rad.
Constants Employed for Determination of Protein
Concentrations--
The extinction coefficients,
1 cm1% at 280 nm, and molecular weights
used for this study were: human protein C (14, 62,000) and APC (14.5, 61,000) (18), rGDPC (13.7, 58,000) (14), human TM (10, 75,000), EPCR
(10, 35,000) (19), bovine (21, 37,000) (15) and human (17.3, 36, 600)
(20) thrombin.
Cell Culture--
Human kidney 293 cells were stably transfected
with human EPCR (referred to as E7 cells) and cultured as described
previously (19). Human umbilical vein endothelial cell(s) (HUVEC) were cultured with endothelial cell growth factor, heparin, and
heat-inactivated bovine calf serum as described (21).
Purification of EPCR and TM from Placenta--
All purification
procedures up to and including immunoaffinity purification were
performed at 4 °C. Two frozen human placentas were thawed, diced,
and passed through a commercial, motor-driven meat grinder (Leeson
Electric Co., Grafton, WI) five times. The ground placenta was
suspended in 1,600 ml of 0.25 M sucrose, 5 mM
benzamidine-HCl, 0.02% NaN3, 5 mM
D-Phe-Pro-Arg chloromethyl ketone (Calbiochem), and 0.02 M Tris-HCl, pH 7.5. Larger particles were partially
disrupted by repeatedly drawing the sample into a 60-ml catheter
syringe followed by rapid sample expulsion. The tissue was collected by
centrifugation at 4,800 × g for 30 min. The pellets
were resuspended and collected by centrifugation as before, except that
the chloromethyl ketone was omitted in the wash buffer and thereafter.
This process was repeated two times. EPCR and TM were extracted from
the washed pellet with 2 volumes of wash buffer made 1% in Triton
X-100 using a Tissumiezer Mark II (Tekmar Company, Cincinnati, OH)
homogenizer. Insoluble material was removed by centrifugation at
22,000 × g for 40 min. The extracts were run quickly
through a 5 × 30-cm column packed with 10 g of swollen
Sephadex G-50 to remove particles and decrease nonspecific binding to
the affinity columns. The filtrate was applied overnight to two
1.5 × 17-cm immunoaffinity columns connected in tandem: an
anti-EPCR 1496 Affi-Gel 10 gel and anti-TM CTM1029 Affi-Gel 10 (5 mg of
mAb/ml of gel). The two columns were washed separately with 2 column
volumes of 0.15 M NaCl, 0.02 M Tris-HCl, pH 7.5 (Tris-buffered saline; TBS), 0.1% Lubrol PX, followed by 4 column volumes of 2 M NaCl, 0.02 M Tris-HCl, pH 7.5, 0.1% Lubrol PX at 1 ml/min.
After washing with 2 additional column volumes of TBS, 0.1% Lubrol PX,
the TM was eluted at 0.5 ml/min with 50% ethylene glycol, 5 mM MES-HCl, pH 6.0, containing 0.1% Lubrol PX. The
protein-containing fractions were pooled and brought to 0.15 M NaCl by the addition of 1.5 M NaCl, 0.2 M Tris-HCl, pH 7.5. This material was applied to a Mono Q
HR5/5 column attached to a fast protein liquid chromatography system
(Pharmacia), washed with 100 ml of the lead buffer, and developed with
a 26-ml linear gradient from 0.1 to 2.0 M NaCl in 0.02 M Tris-HCl, pH 7.5, containing 15 mM OG. For
EPCR, the mAb column was washed with 4 column volumes of TBS containing 15 mM OG at 0.333 ml/min, and the EPCR was eluted with 2 column volumes of 50% ethylene glycol, 15 mM OG, 5 mM MES-HCl, pH 6.0. The protein-containing fractions were
pooled, concentrated, and buffer exchanged into 0.05 M
NaCl, 0.02 M Tris-HCl, pH 7.5, 15 mM OG using a
Centriprep-30 (Amicon). This material was applied to a Mono Q HR5/5
column on a fast protein liquid chromatography system and developed
with a 26-ml linear gradient from 0.05 to 1.0 M NaCl in 15 mM OG, 0.02 M Tris-HCl, pH 7.5. Some minor
contaminants were separated from the EPCR, which was eluted as a single
peak at approximately 0.2 M NaCl.
Purification of EPCR from E7 Cells--
E7 cells were detached
from a cell factory (6,320 cm2) with 2 liters of 0.526 mM EDTA in phosphate-buffered saline (138 mM NaCl, 2.7 mM KCl, 10 mM phosphate, pH 7.4;
Sigma). The cells were centrifuged at 3,500 × g for 30 min. All purification steps were performed at 4 °C. The cell pellets
were extracted for 15 min with 120 ml of 1% Triton X-100, 0.25 M sucrose, 20 mM HEPES-HCl, pH 7.5, 5 mM benzamidine-HCl, 0.02% NaN3, 2 mM EDTA, 1 mM phenylmethanesulfonyl fluoride,
and 1 µg/ml leupeptin. The insoluble material was removed by
centrifugation at 40,000 × g for 30 min, and
particulates were removed with a 0.45 µM filter. The
filtrate was applied to the anti-EPCR mAb 1496 Affi-Gel 10 column (8 ml) at 0.67 ml/min. The column was washed and eluted essentially as
described above for the placental EPCR, except that the volumes were
reduced in proportion to the resin volume. The protein-containing
fractions were pooled, concentrated, and buffer exchanged into TBS
containing 15 mM OG using the Centriprep-30.
Reconstitution of EPCR and TM into Phospholipid
Vesicles--
Purified EPCR and TM were incorporated into phospholipid
vesicles by minor modifications of the method described by Galvin et al. (22). Briefly, 1 mg of phospholipid was dissolved
with 40 µl of 14% OG in TBS. TM (5 µg) and EPCR ranging from 0 to
27.4 µg were added, and the final volume was adjusted to 200 µl
with TBS. [14C]Phosphatidylcholine was included as a
tracer. The samples were dialyzed against three changes, 1 liter each,
of TBS, 0.02% NaN3 for 45 h. The dialyzed sample was
mixed with 0.2 ml of 50% sucrose, overlaid with 0.8 ml of 20%
sucrose, and finally overlaid with 0.2 ml of TBS before centrifugation
at 20 °C in a TLA-100.2 rotor (Beckman) for 16 h at
100,000 × g. 0.2-ml fractions were collected from the
sucrose density gradients. The phospholipid concentration was
determined based on [14C]phosphatidylcholine content
(22). The surface-expressed TM concentration was determined based on
the rate of rGDPC activation and compared with a standard curve with
purified TM as described (22) (also see below). Previous studies have
shown that the activation of GDPC is not influenced by incorporation of
TM into liposomes (22) or by the presence of EPCR (6). EPCR
concentrations were determined with a sandwich ELISA (see below).
Surface-expressed EPCR was assumed to be half of the total associated
with the liposome.
Protein C or GDPC Activation Assay--
Protein C and GDPC
activation rates by liposome-incorporated TM were measured by a
modification of the method of Galvin et al. (22). Briefly,
vesicles containing 20 µg/ml phospholipid and TM or TM and EPCR were
mixed at 37 °C in a 96-well microtiter plate with 0.1 µM protein C or rGDPC in Hanks' balanced salt solution containing 0.1% bovine serum albumin, 3 mM
Ca2+, and 0.6 mM Mg2+. 5 nM human or bovine thrombin was added to initiate
activation. In some cases, 0.1 µM anti-EPCR mAb 1494 or 1 µM annexin V was added before protein C and preincubated
with the vesicles for 5 min at room temperature to block the EPCR or to
block the negatively charged phospholipid surface. The reactions were
stopped at defined time intervals by the addition of 0.20 volume of
antithrombin (1.5 mg/ml) containing 20 mM EDTA, 20 mM HEPES-HCl, pH 7.5. APC and activated rGDPC
concentrations were determined based on their amidolytic activities
toward 0.2 mM Spectrozyme PCa substrate in 0.15 M NaCl, 20 mM HEPES-HCl, pH 7.5. The rates of
substrate cleavage were measured with a Vmax
microplate reader (Molecular Devices). The concentration of enzyme in
these reaction mixtures was determined by comparison with a standard
curve of amidolytic activity versus enzyme concentration
constructed with freshly prepared, fully activated protein C or rGDPC
(22). Under the conditions employed in this study, less than 10% of
the protein C was activated during the assay, and all assays were
linear as a function of time between the initiation and termination of
the assay.
ELISA for EPCR--
The assay was modified from Kurosawa
et al. (23). Microtiter plates were coated overnight at
4 °C with 50 µl of 5 µg/ml anti-EPCR mAb 1494 in 15 mM Na2CO3, 35 mM
NaHCO3, pH 9.6. The remaining steps were performed at room
temperature. The wells were washed three times with TBS containing
0.05% Tween 20 and were blocked with TBS and 0.1% gelatin. The wells
were washed, 50-µl samples in TBS containing 0.05% Tween 20 were
added in duplicate wells, and the plates were incubated for 1 h.
The wells were washed and 50 µl of biotin-conjugated anti-EPCR mAb
1495 (2 µg/ml) or biotin-conjugated anti-EPCR mAb 1,500 (1 µg/ml)
was added. The plates were incubated for 1 h, washed, and 50 µl
of streptavidin-alkaline phosphatase conjugate (1 µg/ml) was added
and incubated for 1 additional h. The wells were washed five times, and
50 µl of 1 mg/ml p-nitrophenyl phosphate substrate was
added. The end point absorbance at 405 nm was read on a
Vmax microplate reader. The standard curve was
linear (r = 0.999) from 0 to 100 ng/ml.
Chondroitinase Treatment of TM and Western Blotting--
TM (0.5 µg/ml) in TBS containing 0.1% Lubrol PX was incubated for 1 h
with 0.5 unit/ml chondroitinase ABC at 37 °C before analysis by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (24). For Western
blot analysis, gels were transferred to polyvinylidene difluoride
membranes (Millipore) in a semi-dry apparatus (Bio-Rad). Membranes were
blocked with non-fat milk, incubated with anti-TM mAb CTM1009, washed,
and incubated with sheep anti-mouse IgG conjugated with horseradish
peroxidase (Amersham Pharmacia Biotech). The membranes were washed and
developed with the ECL substrate (Amersham Pharmacia Biotech).
Preparation and Iodination of mAb Fab Fragments--
Anti-EPCR
mAb 1500 (1.2 mg/ml) and anti-TM mAb CTM1029 (3.3 mg/ml) in
phosphate-buffered saline were added to 7.5 and 30 mM cysteine, respectively. The mAbs were then incubated at 37 °C for 90 min with a 1:50 ratio (w/w) of ficin (Sigma). Iodoacetamide (11.25 mM for mAb 1500 and 45 mM for mAb CTM1029) was
added to inhibit further proteolysis. The digests were buffer exchanged into TBS on a PD-10 column (Amersham Pharmacia Biotech) and purified on
a protein G column (Amersham Pharmacia Biotech). IODO-GEN (Pierce) was
used to radiolabel the Fab fragments according to the manufacturer's protocol. 125I-Labeled Fab fragments were separated from
free iodine on a PD-10 column.
Estimation of EPCR and TM Expression on HUVEC--
Confluent
HUVEC in 24-well plates were washed two times with wash buffer (Hanks'
buffered salt solution containing 0.1% bovine serum albumin, 3 mM Ca2+, 0.6 mM Mg2+).
Binding studies were carried out in reaction mixtures containing medium
199 supplemented with 0.5% bovine serum albumin and 0.1% normal human
IgG in a total volume of 0.2 ml. After incubation of cells with
125I-labeled anti-EPCR (1500) or anti-TM mAb CTM1029 Fab
fragments (30 nM) at 37 °C for 30 min, the cells were
washed rapidly four times with cold wash buffer. The Fab fragments were
then eluted from the cells three times with cold acid buffer (0.15 M NaCl, 20 mM glycine-HCl, pH 2.5).
125 I in the supernatant was measured in an Iso-Data 500
-counter. The number of Fab binding sites was calculated based on
the radioactivity in the supernatant and the specific activity of the
125I-labeled mAb Fab fragments. Nonspecific binding was
determined with a 100-fold excess of the corresponding unlabeled mAb
and was less than 5% of the total counts eluted.
Protein C or GDPC Activation on HUVEC--
HUVEC in 24-well
plates were washed two times with wash buffer and preincubated for 5 min with wash buffer, 0.1 µM 1494, 0.1 µM
CTM1009, or 1 µM recombinant annexin V before the
addition of 0.1 µM protein C or rGDPC. The activation
reactions were initiated by addition of 5 nM bovine
thrombin in a total volume of 0.2 ml. After 30 min at 37 °C, the
reactions were stopped by adding 40 µl of antithrombin (1.66 mg/ml)
to the reactions. The supernatants were transferred to the 96-well
microplate, and the amidolytic activities of activated protein C or
rGDPC were determined toward 0.2 mM Spectrozyme PCa
substrate in 0.15 M NaCl, 20 mM HEPES-HCl, pH
7.5. APC or activated rGDPC concentrations were determined by reference
to a standard curve as described above under "Protein C or GDPC
Activation Assay." All determinations were performed in duplicate.
 |
RESULTS |
Human TM and EPCR were isolated from placenta as described under
"Experimental Procedures." The SDS-PAGE of freshly prepared EPCR is
shown in Fig. 1A and exhibits
one major band with or without disulfide bond reduction. EPCR stored at
4 °C formed some aggregates that were partially eliminated by
disulfide bond reduction (Fig. 1B), consistent with the
aggregation being mediated by the free Cys residues in the
extracellular domain, the transmembrane domain, and the cytosolic tail
of the EPCR (5, 19). EPCR from placenta and EPCR from the transfected
cell line (E7) exhibited similar mobility on SDS-PAGE. The apparent
mass of the EPCR (40 kDa) on SDS-PAGE is approximately twice that
predicted from the amino acid sequence, consistent with the predicted
four N-linked carbohydrate attachment sites on EPCR (5).
Some degradation in the placental EPCR was often observed. Considerably
less degradation was observed in the EPCR isolated from the E7
cells.

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Fig. 1.
SDS-PAGE of the EPCR and TM
preparations. Panel A, EPCR freshly prepared from
placenta (0.1 µg/sample) was analyzed by electrophoresis on 10%
SDS-PAGE without ( ) and with (+) disulfide bond reduction.
BME, -mercaptoethanol. Panel B, EPCR from
placenta and E7 cells and TM from placenta were analyzed by 10%
SDS-PAGE. Lane 1, molecular mass standards; lane
2, EPCR from placenta without reduction; lane 3, EPCR
from E7 cells without reduction; lane 4, TM isolated from
placenta corresponding to the major peak eluted from the Mono Q column.
Lanes 5-7 were equivalent to lanes 2-4 but with
disulfide bond reduction. Panel C,Western blot analysis of
TM from the trailing shoulder of the Mono Q column. TM samples from the
trailing shoulder of the Mono Q column were either treated (+) or not
( ) with chondroitinase ABC. Nonreduced samples were then run on 7.5%
SDS-PAGE.
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Mono Q chromatography of the immunoaffinity-purified TM resulted in a
major peak with a shoulder on the trailing edge. This shoulder was less
than 30% of the TM preparation (data not shown). TM isolated in the
major peak from the Mono Q column (i.e. largely devoid of
chondroitin sulfate, see below) is shown in Fig. 1B. As has
been observed with other human TM preparations from placenta (25),
there are aggregates and some degradation products present in addition
to the major monomeric TM form. Most of these aggregates could be
dissociated by disulfide bond reduction, consistent with the known Cys
residues in the cytoplasmic tail (26-28). The presence of a trailing
shoulder on the main peak from the Mono Q column behavior is indicative
of relatively anionic forms of human TM. This anionic character appears
to be caused by modification with chondroitin sulfate. Western blots of
the trailing fractions revealed a diffuse band that was sharpened after
chondroitinase ABC treatment (Fig. 1C). Rabbit TM has been
shown to contain a chondroitin sulfate moiety that renders the molecule
more anionic, and the proteoglycan form migrates as a diffuse, slower
moving band when analyzed by SDS-PAGE (29). Preliminary data with human
TM preparations are also consistent with this proposal (30).
Antigen levels of TM and EPCR in the Triton X-100 extract of the
placenta were determined by ELISA. Assuming nearly complete extraction,
there were approximately 0.22 mg of EPCR and 0.88 mg of TM per
placenta. The higher TM:EPCR ratio is consistent with the observation
that EPCR is found mostly in large vessels, and TM is in both large and
microvessels, the latter constituting the vast majority of the
endothelium (8).
The availability of purified human TM and EPCR allowed us to
reconstitute the isolated proteins into membrane vesicles using the
detergent dialysis technique (22, 31). Proteins incorporated into the
phospholipid can be separated from free protein by sucrose density gradient centrifugation in which the liposomes are found at the
top of the gradient and the free protein near the bottom. As can be
seen in Fig. 2, placental TM and EPCR
from either tissue culture or from placenta can be incorporated into
vesicles in this fashion. It should be noted that EPCR at either low or
high concentrations does not incorporate as efficiently as TM. This is
possibly because of the presence of two Gly-Gly sequences in the
transmembrane region (5) which would tend to break helix formation at
the beginning and end of the transmembrane region. Placental and
recombinant EPCR were indistinguishable with respect to phospholipid
reconstitution and functional properties and are used interchangeably
in the following experiments. When the EPCR that did not incorporate
into phospholipid was isolated and the reconstitution protocol was
repeated, the EPCR again failed to incorporate into the liposomes (data
not shown). The molecular basis for this behavior is currently under
investigation.

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Fig. 2.
SDS-PAGE analysis of EPCR and TM
incorporation into liposomes. Fractions from a sucrose density
gradient separating free and phospholipid incorporated EPCR and TM were
subjected to 10% SDS-PAGE. Left panel, human placental EPCR
and TM incorporation into phosphatidylcholine vesicles with an EPCR:TM
ratio of 0.6 (w/w) in the reconstitution mixture; right
panel, EPCR isolated from E7 cells and placental TM were
reconstituted into phosphatidylcholine vesicles at a 5.6:1 w/w
ratio.
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The EPCR concentration dependence of protein C activation was examined
by adjusting the EPCR concentration during the reconstitution. It was
possible to vary the EPCR:TM ratio from 1:1 to 14.2:1. As the EPCR
ratio increased, the rate of protein C activation increased (Fig.
3). To separate the EPCR effects on the
activation reactions from variations in TM incorporation, we took two
separate approaches. First, we determined the activation rate of rGDPC. Without the Gla domain, protein C can be activated by the soluble thrombin-TM complex, but the activation rate is not influenced by
reconstitution into neutral or negatively charged phospholipid (22),
and the molecule cannot bind to EPCR (5). Second, activation rates in
the presence of anti-EPCR mAb 1494 which blocks protein C binding to
the EPCR were determined (6). The results indicate that the increased
rates of protein C activation observed when EPCR is incorporated are a
direct result of the presence of EPCR rather than changes in the TM
concentration in the liposomes. The activation rate of rGDPC was
relatively constant between preparations and was not influenced by EPCR
density (Fig. 3). The antibody blocked protein C activation rates on
phosphatidylcholine liposomes containing EPCR and TM to near those
observed with rGDPC (Fig. 3), but annexin V had no influence in this
system (data not shown). Inclusion of phosphatidylserine,
phosphatidylethanolamine, or a combination of the phospholipids in the
vesicles accelerated protein C activation in the absence of EPCR. This
acceleration was inhibited by annexin V (Fig. 3) but not by the
anti-EPCR mAb 1494 (data not shown). Incorporation of TM into vesicles
with or without EPCR had little if any effect on the affinity of TM for
thrombin (Kd(app)
2.5 nM),
consistent with previous studies that failed to demonstrate a
phospholipid influence on the affinity of thrombin for TM (7, 22).

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Fig. 3.
Influences of EPCR concentration or
negatively charged phospholipids on protein C and rGDPC activation
rates. EPCR and TM were reconstituted into phosphatidylcholine
vesicles (TM/PC) at the EPCR:TM ratios indicated. Gray
bars, activation rate of rGDPC; black bars, activation
rate of protein C; white bars, activation rate of protein C
in the presence of 0.1 µM inhibitory anti-EPCR mAb 1494. TM was also incorporated into vesicles containing 20%
phosphatidylserine, 80% phosphatidylcholine (TM-PS/PC),
40% phosphatidylethanolamine, 60% phosphatidylcholine
(TM-PE/PC), or 20% phosphatidylserine, 40%
phosphatidylethanolamine, 40% phosphatidylcholine
(TM-PS/PE/PC). Gray bars, activation rate of
rGDPC; black bars, activation rate of protein C;
hatched bars, activation rate of protein C in the presence
of 1 µM annexin V. TM concentrations on these liposomes
were (from left to right) 0.078, 0.124, 0.28, 0.444, 0.142, 0.172, and 0.218 nM. EPCR surface
concentrations were 1.11, 1.07, and 0.28 nM. The
phospholipid concentration was 20 µg/ml in all cases. The initial
rates of protein C activation were performed at 37 °C as described
under "Experimental Procedures." All assays were performed in
duplicate, and all errors were within 5%.
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Blocking protein C binding to the EPCR on cell surfaces reduces protein
C activation rates primarily by increasing the Km (6). When the rate of activation as a function of protein C concentration was examined (Fig. 4), it
was apparent that the affinity for protein C was increased by either
EPCR or the presence of negatively charged phospholipids. Fitting the
data to Michaelis-Menten kinetics indicated that on
phosphatidylcholine-containing vesicles, the Km was
2.17 ± 0.13 µM without EPCR. With EPCR present, the
data could not be fit well to Michaelis-Menten kinetics (Fig. 5A) but did fit well to a
two-site model with a tight site, Km = 20 ± 15 nM, and a weak site, Km = 3.3 ± 1.7 µM (Fig. 5B). Increasing the EPCR:TM
ratios from 8.6:1 to 14:1 increased the percentage of tight sites from
14 to 23%. The necessity for a two-site model to fit the data was only
observed with the EPCR-containing liposomes. Michaelis-Menten kinetics
fit the data well even when liposomes contained TM,
phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine
(Fig. 5C) (Km = 0.89 ± 0.22 µM). The Kcat values for all
reaction mixtures were similar. For phosphatidylcholine liposomes
containing EPCR and TM (ratio = 14.2:1), the
Kcat was 11.1 ± 1.7 mol of APC/min/mol of
TM. For liposomes containing TM, 40% phosphatidylethanolamine, 20%
phosphatidylserine, and 40% phosphatidylcholine the
Kcat was 11.1 ± 0.8 mol of APC/min/mol of
TM. For liposomes containing TM and phosphatidylcholine, the Kcat was 8.0 ± 0.2 mol of APC/min/mol of
TM. The Kcat values observed here are much lower
than those observed with rabbit TM, about 250 mol/min/mol of TM (32),
but are comparable to human placental TM in solution (10 mol/min/mol of
TM)(25) and human placental TM reconstituted into negatively charged
vesicles (8 mol/min/mol of TM) (7) reported previously.

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Fig. 4.
Influence of EPCR and negatively charged
phospholipid on protein C activation as a function of protein C
concentration. Phosphatidylcholine vesicles containing TM and EPCR
( ) had an EPCR:TM ratio of 8.6:1, 0.124 nM TM and 20 µg/ml phosphatidylcholine in the activation mixture. The TM and
phosphatidylcholine vesicles devoid of EPCR ( ) had a TM
concentration of 0.44 nM and phosphatidylcholine
concentration of 20 µg/ml in the activation mixture. The 20%
phosphatidylserine, 40% phosphatidylethanolamine, 40%
phosphatidylcholine liposomes containing TM ( ) had a TM
concentration of 0.218 nM and a phospholipid concentration
of 20 µg/ml in the activation mixture. The initial rates of protein C
activation were determined at the protein C concentrations indicated on
the x axis. The inset expands the graph to focus on
activation rates at low protein C concentrations.
|
|

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Fig. 5.
Analysis of the protein C concentration
dependence of activation. Panel A, the rate of protein
C activation on phosphatidylcholine vesicles containing a 14:1 EPCR:TM
ratio (TM = 0.078 nM) was examined as a function of
increasing substrate concentration, and the data were fit to
Michaelis-Menten kinetics using the Ultrafit program for Macintosh from
Biosoft. Panel B, the data from panel A were fit
to a two-site model where v = (VmaxA·C)/(Km(A)+C) + (VmaxB·C)/(Km(B) + C) where v is the observed velocity,
VmaxA is the Vmax of the
high affinity sites, VmaxB is the
Vmax of the low affinity sites, C is the
concentration of protein C, Km(A) is the
Km of the high affinity site, and
Km(B) is the Km of the low
affinity sites. The data were fit using the Ultrafit program.
Panel C, the rate of protein C activation on vesicles
containing 20% phosphatidylserine, 40% phosphatidylethanolamine, 40%
phosphatidylcholine, and 0.218 nM TM was studied as a
function of protein C concentration, and the data were fit to
Michaelis-Menten kinetics using the Ultrafit program.
|
|
To ascertain whether the absolute density of the EPCR and TM on the
liposome surface plays an important role in EPCR acceleration, we
varied the amount of both the EPCR and TM incorporated into the
liposomes by changing the phospholipid:protein ratio during the
reconstitution. The ratios of protein C to rGDPC activation rates were
then used as a monitor of EPCR function. As the density of the
receptors on the vesicle surface decreased (Fig.
6, highest density on the
left, lowest on the right), the selective
enhancement of protein C activation over rGDPC activation decreased
progressively. The selective enhancement of protein C over rGDPC
activation rates was eliminated by the anti-EPCR mAb 1494 (hatched bars), indicating that the loss of this property
was a result of the loss of EPCR function and not a property of altered
TM:phospholipid ratios.

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|
Fig. 6.
Influence of EPCR and TM surface
density on the protein C activation. TM (5, 2.5, 1.25, and 0.625 µg) was reconstituted into 1 mg of phosphatidylcholine-containing
vesicles with correspondingly decreasing amounts of EPCR (13.7, 6.85, 3.43, and 1.72 µg). The mixtures were brought to 200 µl and the
reconstituted liposomes isolated and characterized as described under
"Experimental Procedures." The concentrations of phospholipid
([PL]) and TM are indicated on the figure, as are the
EPCR:TM ratios of each sample. The ratio of the rate of protein C
activation to the rate of rGDPC activation was taken as a monitor of
EPCR effectiveness. These assays were performed in the presence
(hatched bars) and absence (solid bars) of the
inhibitory anti-EPCR mAb 1494 (0.1 µM).
|
|
To determine the relationship between the reconstituted system and
activation over HUVEC, we estimated the TM and EPCR concentrations on
the cells using 125I-radiolabeled mAb Fab fragments to
EPCR and TM. When binding was performed as described under
"Experimental Procedures," we estimated about 700,000 EPCR
molecules and 100,000 TM molecules/HUVEC. This estimate of surface TM
expression on HUVEC is slightly higher than the earlier estimate of
30,000-55,000 based on intact mAb binding (33). Blocking protein C
binding to EPCR with anti-EPCR mAb 1494 decreased the activation rate
on HUVEC 9-fold (Fig. 7). In contrast,
annexin V, which effectively inhibits protein C activation on
negatively charged vesicles (Fig. 3), had little influence on protein C
activation over quiescent HUVEC. Similar rates of protein C activation
were observed over HUVEC (2.7 mol of APC/min/mol of TM) and
phosphatidylcholine vesicles with an 8.6:1 EPCR:TM ratio (3.2 mol of
APC/min/mol of TM). Thus, the contribution of the HUVEC surface to
protein C activation can be adequately accounted for with the simple
system containing phosphatidylcholine, EPCR, and TM. Negatively charged
phospholipids appear to contribute little to this process.

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Fig. 7.
Protein C activation by HUVEC is effectively
inhibited by an anti-EPCR mAb but not by annexin V. Protein C or
rGDPC activation by HUVEC was measured as described under
"Experimental Procedures." rGDPC activation rates were similar to
the activation rate of protein C in the presence of 0.1 µM mAb 1494 (anti-EPCR+PC) and much slower
than protein C in the absence of the antibody (PC).
Recombinant annexin V (1 µM) was not an effective
inhibitor of protein C activation (rAn V+PC). Inhibiting
thrombin binding to TM with anti-TM mAb CTM1009 (0.1 µM)
almost eliminated protein C activation (anti-TM +PC). The TM
concentrations on the cell surface were estimated using
125I-Fab fragments and corresponded to approximately
100,000 sites/endothelial cell. All determinations were performed in
duplicate.
|
|
 |
DISCUSSION |
Previous studies demonstrated that inhibition of EPCR-protein C
interaction could reduce protein C activation rates (6) and that
coexpression of EPCR and TM increased the TM-dependent protein C activation rate (10). Both of these studies demonstrate involvement of EPCR in protein C activation, but neither rules out a
requirement for another cellular component in this process. The present
study demonstrates that EPCR and TM are the only components required to
enhance protein C activation. The protein C activation rates by HUVEC
with EPCR:TM ratios of approximately 7:1 were similar to those obtained
with EPCR and TM reconstituted into phosphatidylcholine vesicles at
ratios of 8.6:1, i.e. 2.7 versus 3.2 mol/min/mol
of TM-thrombin complex. In addition, the rates of protein C activation compared with rGDPC activation were about 9-fold greater on both endothelium and phosphatidylcholine liposomes containing an EPCR:TM ratio of 8.6:1. The ability to inhibit protein C activation over quiescent HUVEC with the anti-EPCR mAb, but not with annexin V, argues
strongly that the protein C interaction with the cell surface that
augments activation is mediated primarily if not exclusively by EPCR
rather than by negatively charged phospholipids. This provides
selectivity for protein C activation because the other vitamin
K-dependent proteins would compete effectively for the phospholipid surface and inhibit protein C activation, but these factors do not compete effectively for binding to EPCR (34).
The enhancement observed with EPCR is small compared with other
coagulation cofactors that can increase activation rates thousands of
fold (35). One might question whether this relatively small change in
activation rate is biologically important. The answer would appear to
be yes because it is now well established that even a 2-fold reduction
in protein C concentration can result in an increased risk of venous
thrombosis (36). This propensity toward thrombosis is almost certainly
the result of a reduction in protein C activation rates associated with
the decrease in circulating protein C concentration rather than
consumption because very little protein C is activated tonically (37).
Therefore, increases in protein C activation rates of 9-fold or greater
as observed here are likely to play an important role in regulating the
blood coagulation process in large vessels. The much higher effective
TM concentration in the microcirculation can probably generate
sufficient APC to prevent microvascular thrombosis as long as the
protein C concentration remains high. Consistent with this hypothesis,
partial reductions of protein C concentration are usually associated
with large vessel thrombosis, and very low levels are associated with
microvascular thrombosis (36, 38).
Immunohistochemical studies revealed previously that EPCR expression is
much greater on large vessel endothelium, particularly large arteries,
and decreases until it is nearly undetectable in most capillary
endothelial beds (9). The observation that protein C activation rates
rise with increasing concentrations of EPCR provides the first evidence
that there is an EPCR concentration dependence to protein C activation
even when the EPCR concentration exceeds that of TM. The most likely
explanation for the EPCR concentration dependence (lack of apparent
saturation) observed in this study is that there is little or no direct
interaction between TM and EPCR. Otherwise, one would predict
saturation at low integer ratios of EPCR to TM, especially given that
the local concentrations expressed in two dimensions are quite high for
these reconstituted proteins. The conclusion that there is a lack of
appreciable affinity between EPCR and TM is bolstered by the
observation that as the phospholipid to protein ratio increases,
thereby decreasing the surface density of EPCR and TM, the efficiency
of the thrombin-TM complexes is decreased. This is true even though the
EPCR to TM ratios remained essentially constant (Fig. 6).
Analysis of the protein C concentration dependence over liposomes
containing EPCR and TM revealed a complex pattern that did not fit
simple Michaelis-Menten kinetics, whereas activation over liposomes
devoid of EPCR fit Michaelis-Menten kinetics very well. The curve with
EPCR-containing vesicles was consistent with a population of activation
complexes that were high affinity for protein C and another population
that was low affinity (see Figs. 4 and 5). The high affinity sites
(Km
20 nM) are considerably below
the plasma protein C concentration (65 nM), whereas the low
affinity sites (Km
2-3 µM) are
considerably above this value. The high affinity Km
is similar to the Kd for protein C/APC binding to
EPCR estimated in previous studies (30-50 nM) (5). One
interpretation of the apparent two classes of sites is that the higher
affinity sites involve the EPCR-protein C complex, but the low affinity
sites represent catalysis by the free thrombin-TM complex. Further
analysis of the complex kinetic pattern was not attempted because it
would require knowledge of lateral mobility and the off rate of the APC
from EPCR.
The present study demonstrates that the EPCR concentration plays a
major role in determining protein C activation. Up-regulation of EPCR
by thrombin (12) or down-regulation of EPCR expression by cytokines (5)
or proteolytic attack (39, 40) would, based on the present study,
contribute directly to protein C activation and hence serve to modulate
the critical control of the blood clotting process.
 |
ACKNOWLEDGEMENTS |
We thank Pierre Neuenschwander for assistance
in the kinetic analyses, Steve Carpenter for the cell culture work,
Clendon Brown for placenta protein purification assistance, and Jeff
Box for preparation of the figures.
 |
FOOTNOTES |
*
This research was supported by NHLBI, National Institutes of
Health, Grant P01 HL54804.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.
**
Investigator with the Howard Hughes Medical Institute. To whom
correspondence should be addressed: Howard Hughes Medical Institute, Oklahoma Medical Research Foundation, 825 NE 13, Oklahoma City, OK
73104. Tel.: 405-271-7571; Fax: 405-271-3137; E-mail:
Charles-Esmon{at}omrf.ouhsc.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
TM, thrombomodulin;
APC, activated protein C;
EPCR, endothelial cell protein C receptor;
rGDPC, recombinant Gla-domainless protein C;
mAb, monoclonal antibody;
OG, octyl-
-D-glucopyranoside;
HUVEC, human umbilical
vein endothelial cell(s);
TBS, Tris-buffered saline;
MES, 2-(N-morpholino)ethanesulfonic acid;
ELISA, enzyme-linked
immunosorbent assay;
PAGE, polyacrylamide gel electrophoresis.
 |
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