Protein C is an important regulatory mechanism of blood coagulation. Protein C functions as
an anticoagulant when converted to the active serine protease form on the endothelial cell surface. Thrombomodulin (TM), an endothelial cell surface receptor specific for thrombin, has
been identified as an essential component for protein C activation. Although protein C can be
activated directly by the thrombin-TM complex, the conversion is known as a relatively low-affinity reaction. Therefore, protein C activation has been believed to occur only in microcirculation. On the other hand, we have identified and cloned a novel endothelial cell surface receptor (EPCR) that is capable of high-affinity binding of protein C and activated protein C. In
this study, we demonstrate the constitutive, endothelial cell-specific expression of EPCR in
vivo. Abundant expression was particularly detected in the aorta and large arteries. In vitro cultured, arterial endothelial cells were also found to express abundant EPCR and were capable of
promoting significant levels of protein C activation. EPCR was found to greatly accelerate protein C activation by examining functional activity in transfected cell lines expressing EPCR
and/or TM. EPCR decreased the dissociation constant and increased the maximum velocity
for protein C activation mediated by the thrombin-TM complex. By these mechanisms,
EPCR appears to enable significant levels of protein C activation in large vessels. These results
suggest that the protein C anticoagulation pathway is important for the regulation of blood coagulation not only in microvessels but also in large vessels.
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Introduction |
Protein C pathway is an indispensable regulatory mechanism of blood coagulation, since deficiencies in this
pathway lead to thrombosis (1, 2). Protein C is a
-carboxyglutamic acid containing protein (3) and circulates as a zymogen form of serine protease (4). Protein C functions as
an anticoagulant when converted to its active form by
thrombin (5), and activation of protein C was demonstrated to be greatly enhanced on the endothelial cell surface (6). Thrombomodulin (TM)1 has been identified as an
essential component on the endothelial cell surface (7, 8),
and direct activation of protein C by the thrombin-TM
complex has been demonstrated. However, the activation
by the complex was observed as a relatively low-affinity reaction and the Kd value was calculated as 0.7-1.0 µM (9-
11), which is 15 times higher than that of the protein C
concentration (65 nM) in the circulation (12). Under these
conditions, protein C activation appears unlikely to occur
under normal physiological conditions. One of the proposed explanations for this has been that protein C activation mediated by the thrombin-TM complex occurs only
in the microcirculation because of the greater ratio of endothelial cell surface area to blood volume (13).
On the other hand, we (14) and others (15) found that
protein C and activated protein C (APC) bound to cultured endothelial cells with a relatively high affinity (Kd = 30 nM). By expression cloning, we identified a novel endothelial cell surface receptor that is capable of protein C-APC
binding in the presence of physiological concentrations of
calcium (14) and magnesium (16). We designated this molecule as an endothelial cell protein C-APC receptor (EPCR). EPCR is a type 1 transmembrane glycoprotein containing
two domains in the extracellular region that are homologous to the
1 and
2 domains of CD1/MHC class 1 molecules (17). The highly conserved structural features and
ligand binding function of EPCR that are found between
species suggests an important physiological function for the
receptor molecule (18). In in vitro cultured human cells, significant levels of EPCR expression has been demonstrated only in human umbilical vein endothelial cells (14).
In this study, we demonstrate in vivo expression of EPCR
by immunohistochemical analysis. The abundant expression of EPCR was found on the endothelial cells of the
aorta where protein C activation has been considered unlikely to occur. In addition, we found that protein C
activation mediated by the thrombin-TM complex was
promoted as a relatively high-affinity reaction in the presence of EPCR.
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Materials and Methods |
Cells.
Human primary venous endothelial cells (VECs) and
arterial endothelial cells (AECs) were obtained from Cell Systems
(Kirkland, WA) and maintained according to the manufacturer's
protocol. Human kidney 293 cells (CRL 1573; American Type
Culture Collection, Rockville, MD) were maintained in DMEM
containing 10% fetal bovine serum. Stable transfected cell lines of
293 cells expressing human EPCR and/or TM were established
as follows. Cells were transfected with a human EPCR cDNA
construct in a mammalian expression vector, pEF-BOS (19),
and/or a human TM cDNA (a gift from J.F. Parkinson, Lilly Research Lab., Indianapolis, IN) constructed in a mammalian expression vector pBK-EF (a gift from T. Fujimoto, Hiroshima
University, Hiroshima, Japan) by the calcium/phosphate method
as described (14). Cell lines were established by G-418 selection,
followed by subcloning. T2 cells are positive for TM. ET1 cells
and ET2 cells are positive for both EPCR and TM. Establishment
of these cell lines was done in the Oklahoma Medical Research
Foundation (Oklahoma City, OK) under the support of C.T. Esmon
(Oklahoma Medical Research Foundation). Negative control N1
cells and EPCR positive E7 cells were established as described (16).
Histology.
Sections (4 µm) of human aorta and lung were fixed
in cold acetone, paraffin embedded processed, and then stained with
JRK-1, an anti-EPCR mouse monoclonal antibody, or CTM1009,
an anti-TM mouse monoclonal antibody (a gift from C. Carson,
Oklahoma Medical Research Foundation, Oklahoma City, OK).
Antigen expression was then detected using biotinylated anti-
mouse Ig, streptavidin-conjugated horseradish peroxidase, and
3,3'-diaminobenzidine as previously described (20).
Flow Cytometry.
Cells were harvested and stained with JRK-1
or CTM1009 as described (14). The antigen expression was detected on a FACScan® flow cytometer (Becton Dickinson,
Sunnyvale, CA) by using FITC-conjugated goat anti-mouse IgG
(Southern Biotechnology Associates, Inc., Birmingham, AL).
Living cells were gated by staining with propidium iodide. Data
analysis was performed by using the Win MDI program (J. Trotter, Scripps Research Institute, La Jolla, CA).
Protein C Activation Assay.
Monolayers of AECs or VECs
were formed by overnight culture in 48-well microplates coated
with gelatin. After washing with PBS containing 0.5 mM EDTA,
the cells were incubated with various concentrations of human
protein C in 100 µl of HBSS containing 0.1% BSA in the absence or presence of human thrombin (0.1 U final concentration). The plates were incubated at 37°C for 30 min. To stop the
reaction, antithrombin III, at a final concentration of 1 µg/ml,
and EDTA, at a 5 mM final concentration, were added and the
plates were incubated for a further 5 min. Aliquots were transferred to 96-well microplates and a chromogenic substrate, S-2366
(AB Kabi Diagnostica, Stockholm, Sweden), was added at a final
concentration of 200 µM. The catalytic rate of the substrate was
measured by absorbance at 405 nm by using a Vmax kinetic microplate reader (Molecular Devices Co., Sunnyvale, CA). Generated
APC was estimated by using a standard curve of purified APC.
The same experiments were also carried out by using monolayers
of transfected human 293 cells (see above) formed in microplates
coated with poly-L-lysine (Sigma Chemical Co., St. Louis, MO).
The Kd and Vmax values were calculated using the Hyper program
(J.S. Easterby, University of Liverpool, Liverpool, UK).
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Results and Discussion |
In a previous study, we demonstrated that the message
expression of EPCR was demonstrable only with HUVECs among in vitro cultured human cells (14). The messenger RNA was also detectable in normal tissues by Northern blot analysis (data not shown). Abundant expression
was detected in heart and placenta, and the message was
also present in lung, kidney, and pancreas, although brain
was negative for the message. To investigate the detailed expression pattern of EPCR, we performed immunohistochemical analysis of human tissues using an anti-EPCR
monoclonal antibody and anti-EPCR goat Ig (16). Constitutive, endothelial cell-specific expression of EPCR was
demonstrated with these antibodies. The most abundant expression of EPCR was detected on the endothelial cells
of the aorta (Fig. 1 A). Moderate expression was also detected on the endothelial cells of relatively large-sized vessels (data not shown). On the other hand, little or no expression could be detected in microvessels such as the lung
capillaries (Fig. 1 C).

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Fig. 1.
Immunoperoxidase
staining of EPCR. Sections of human aorta (A) and lung (C) were
stained with an anti-EPCR monoclonal antibody, JRK-1, by using
biotinylated anti-mouse goat Ig
and avidin-linked peroxidase. In
control experiments, the sections
were stained with the same reagents, but without the first antibody (B and D). The endothelial
cells in aorta were positive for the
antigen (A), and those in lung capillary were negative (C).
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We also examined serial sections using an anti-TM monoclonal antibody. TM was expressed widely in both arterial
and venous vessels as described (21), and strong expression
was detected in the capillaries (data not shown). These results indicated that EPCR and TM coexpress on the endothelial cells in vessels with relatively large size. Coexpression of EPCR and TM was also demonstrated by the flow
cytometer analysis using in vitro cultured endothelial cells
(Fig. 2). Strong expression of EPCR was detected on cultured AECs. EPCR was also detectable in cultured VECs;
however, the expression level was several times lower than
that in the AECs. TM was expressed on both AECs and
VECs. In contrast to EPCR, strong expression of TM was
detected on VECs and a lower and heterogeneous expression was detected on AECs.

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Fig. 2.
Surface expression of EPCR and TM on cultured endothelial
cells. In vitro cultured AECs (top) and VECs (bottom) were stained with
JRK-1 (an anti-EPCR monoclonal antibody, left) or CTM1009 (anti-TM
monoclonal antibody, right). The antigens expression was detected by
flow cytometer analysis using FITC-conjugated anti-mouse Ig. The x-axis
represents the fluorescence intensity (log scale) and the y-axis represents
relatively cell number.
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Since the dissociation constant between protein C and
EPCR is 30 nM, which is approximately half of the blood
concentration of protein C, the majority of EPCRs exposed to the blood stream should be holding protein C under physiological conditions. These findings encouraged us
to reinvestigate the mechanism of protein C activation,
which has been believed to be mediated by only the thrombin-TM complex. We compared activities of protein
C activation by AECs and VECs, which express different
amounts of EPCRs and TM on their surface (Fig. 2). In
both cases, protein C was converted to APC only in the
presence of thrombin. Interestingly, promotion of protein
C activation by AECs was comparable to VEC-mediated activation (Fig. 3), despite the lower level of TM expression (Fig. 2). In addition, activation of protein C on AECs
was observed as a relatively high-affinity reaction with a Kd
value of 125 ± 72 nM, suggesting contribution by the
high-affinity protein C receptor to the reaction. The Kd
value for the reaction by VECs was slightly lower (162 ± 54 nM) than that of AECs. This could be due to the low
level of expression of EPCR on VECs.

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Fig. 3.
Protein C activation
by cultured endothelial cells.
Protein C was incubated with
venous (A and B) or arterial (C
and D) endothelial cells in the
presence of thrombin, and generated APC was measured. The
y-axis represents velocity of APC
generation (pM/min) in the
presence of indicated concentrations of protein C (A and C).
The Vmax and Kd values were
calculated by the Eadie-Hostee
plot (B and D).
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To analyze in detail the activation mechanism, we established transfected human kidney 293 cell lines that express
receptor molecules. The 293 cells are negative for EPCR
and TM. Mock-transfected N1 cells are used as a negative
control, E7 cells are EPCR positive as described previously
(16), T2 cells are positive for TM alone, and ET1 and ET2
are dual-positive cell lines expressing both EPCR and TM.
Stable antigen expression was demonstrated by flow cytometer analysis as shown in Fig. 4. Protein C was incubated with these cell lines in the presence of thrombin and
the subsequently generated APC was measured. Thrombin-mediated protein C activation was not detectable on the
negative control N1 cells. EPCR-positive E7 also could
not promote activation. On the other hand, thrombin-dependent protein C activation was detected with cells
positive for TM. T2 cells promoted activation only in the
presence of thrombin. The activation was completely inhibited by a functional blocking anti-TM monoclonal antibody, CTM1009 (Fig. 5 A). These results indicated that
activation of protein C on T2 cells was mediated by the
thrombin-TM complex. However, the conversion rate was
quite low when the rate per cell was calculated. In contrast,
dramatic activation was demonstrated with EPCR/TM
dual-positive cells (Fig. 5 A). Five to six times more activation was detected in the case of dual-positive cells as compared with cells positive for TM alone.

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Fig. 4.
Surface expression EPCR and TM on transfected cell lines.
Cells were stained with CTM1009 (left) or JRK-1 (right) monoclonal antibodies. The antigen expression was analyzed by flow cytometer analysis.
N1 is a negative control cell line. E7 was established from cells transfected
with EPCR. T2 was from TM-transfected cells. ET1 and ET2 were from
dual-transfected cells with both EPCR and TM.
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Fig. 5.
Protein C activation by transfected
cells. Monolayers of indicated cells were incubated with 300 nM of protein C and 0.1 U of
thrombin in the absence or presence of 10 µg/
ml of CTM1009, and then the generated APC
was measured (A). ET1, T2, and N1 cells were
incubated with indicated concentrations of protein C in the presence of thrombin, and the
generated APC was measured (B). The Kd and
Vmax values of protein C activation by ET1 cells
(C) or T2 cells (D) were calculated by the
Eadie-Hostee plot.
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We next compared APC generation by T2 cells and ET1
cells in the presence of increasing protein C concentration
(Fig. 5 B). Protein C activation by T2 cells was greatly dependent on the protein C concentration. On the other
hand, dual positive ET1 cells could promote significant activation, even at the lowest concentration of protein C. The Kd value of the reaction was calculated as 869 ± 396 nM for T2 cells (Fig. 5 D) and 140 ± 43 nM for ET1 cells
(Fig. 5 C). The low-affinity reaction by cells positive for
TM alone and the high-affinity reaction by dual-positive
cells were also demonstrated with the other cell lines (data
not shown). The Kd value obtained using T2 cells was
identical with that calculated in a reconstitution experiment using solubilized TM in phospholipid membranes
(11). In contrast, the Kd value for ET1 cells was almost
identical with that of AEC (Fig. 3). Therefore, EPCR/TM dual-positive cells, but not cells positive for TM alone,
might mimic the protein C activation mechanism observed
in in vitro cultured endothelial cells. TM appears to be an
essential component for activation even in the presence of
EPCR, because complete inhibition of protein C activation could be demonstrated again using the anti-TM monoclonal antibody (Fig. 5 A).
To analyze the function of EPCR on endothelial cells,
we established several functional blocking monoclonal antibodies against EPCR (Ye X., N. Tsuneyoshi, K. Fukudome, and M. Kimoto, unpublished data). One of these,
RCR-252 (rat IgG1), was found to inhibit APC binding to
EPCR-expressing cells (Fig. 6 A). RCR-252 also inhibited
protein C activation mediated not only by EPCR/TM
dual-transfected ET1 cells (Fig. 6 B), but also by primary
cultured AECs (Fig. 6 C) in the same manner. The inhibitory effect of RCR-252 was specific for EPCR function
since protein C activation mediated by TM single-positive
T2 cells was not affected (Fig. 6 B). Therefore, EPCR on
cultured AECs appears to function for protein C activation. Inhibition of protein C activation by RCR-252 was only
partial (~60%), in sharp contrast with the complete inhibition by anti-TM antibody (Fig. 6 C). This would be partially due to the fact that EPCR is a regulatory, but not an
essential, component for the catalytic reaction in such a
way that EPCR amplifies dramatically the reaction mediated by the thrombin-TM complex (Fig. 5). In addition,
binding of APC to ET1 cells by RCR-252 is not blocked completely. Approximately 10% of binding still remains,
even in the presence of the highest concentration of the
antibody (Fig. 6 A). Therefore, actual contribution of
EPCR on protein C activation would be greater than that
shown in Fig. 6.

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Fig. 6.
Effects of anti-EPCR monoclonal antibodies on APC binding and protein C activation. ET1 cells were treated with indicated concentrations of a functional blocking anti-EPCR monoclonal antibody,
RCR-252 (closed circles) or a nonblocking RCR-122 (open circles), and
then incubated with 300 nM of APC. After washing, amidolytic activity
of bound APC was measured by using S-2366 and indicated as the maximum velocity (A). ET1(closed circles), T2 (closed squares), and N1 (closed triangles) cells were treated with indicated concentrations of RCR-252. As a
negative control, RCR-122 was used (open circles). Cells were then incubated with 300 nM of protein C and 0.1 U of thrombin. Generated APC
was measured and indicated as described above (B). After incubation of
AECs with RCR-252 (circles), CTM1009 (squares), or RCR-252 (triangles), APC generation was measured (C).
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Since the presence of EPCR reduced the Kd value for
protein C activation mediated by the thrombin-TM complex, EPCR appears to concentrate protein C on the endothelial cell surface. Protein C appears to change conformation when it binds to EPCR because the velocity of the
catalytic reaction mediated by the thrombin-TM complex
increased in the presence of EPCR. Although EPCR can
bind protein C with high affinity, the binding has been
demonstrated as a reversible reaction (15). Therefore, bound
protein C should be replaced continuously in circulation.
By these mechanisms, protein C activation appears to be
promoted effectively on the endothelial cell surface. Whether
EPCR and TM exist as a complex on the endothelial cell
surface or the complex is formed after binding of protein C
to EPCR remains to be investigated.
Clinical studies related to the protein C pathway have
been mainly focused on thrombosis in microvessels, since
until now protein C activation has been considered to be
restricted to the microcirculation (Fig. 7 A). However, in
this study, we have demonstrated a novel mechanism for
APC generation involving EPCR (Fig. 7 B). The high-affinity protein C receptor is expressed in large vessels in
which protein C activation has been believed unlikely to
occur. EPCR appears to enable significant levels of protein
C activation in large vessels under physiological conditions.
Generation of APC might function as an important regulatory mechanism for blood coagulation in large vessels. In
fact, some clinical reports of arterial thrombosis appear to
be related to defects in the protein C pathway (22). In
the microvessels, we could not detect EPCR. Whether the
physical condition of increasing the endothelial cell surface
area to blood volume make up the low-affinity reaction or
another unidentified molecule is involved in the activation remains to be investigated.

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Fig. 7.
Hypothetical models for protein C activation on
the endothelial cell surface. In
microvessels, EPCR is not detectable. In the absence of
EPCR, protein C activation mediated by the thrombin-TM
complex is a relatively low-affinity reaction. For significant levels
of protein C activation to occur,
an increasing ratio of endothelial
cell surface area to the blood volume is required (A). In the large
vessels, the high-affinity binding
of protein C to EPCR on the
endothelial cells is a critical step
for activation (B). After binding
to EPCR, the structure of protein C is modulated to enable
rapid conversion to the active
form.
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Address correspondence to Kenji Fukudome, Department of Immunology, Saga Medical School, 5-1-1 Nabeshima, Saga 849, Japan. Phone: 81-952-34-2256; Fax: 81-952-34-2049; E-mail: fukudome{at}post.saga-med.ac.jp
The authors thank Drs. K. Miyake and Y. Kamikubo for their helpful discussion; Dr. F. Nestel for critical
reading of the manuscript; and S. Chen, F. Mutho, and C. Brown for technical support.
This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan to
K. Fukudome and M. Kimoto, and from the Ryouichi Naitoh Foundation for Medical Research to K. Fukudome.
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