(Circulation. 1997;96:3633-3640.)
© 1997 American Heart Association, Inc.
Articles |
Human Protein C Receptor Is Present Primarily on Endothelium of Large Blood Vessels
Implications for the Control of the Protein C Pathway
From the Departments of Pathology (Z.L., A.M., C.T.E.) and Biochemistry and Molecular Biology (C.T.E.), University of Oklahoma Health Sciences Center; the Cardiovascular Biology Research Program (F.B.T., C.T.E.), Oklahoma Medical Research Foundation; and the Howard Hughes Medical Institute (G.F., C.T.E.), Oklahoma City, Okla.
Correspondence to Charles T. Esmon, PhD, Howard Hughes Medical Institute, Oklahoma Medical Research Foundation, 825 NE 13, Oklahoma City, OK 73104. E-mail Charles-Esmon{at}mrf.uokhsc.edu
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Abstract |
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Background The protein C anticoagulant pathway is critical to the control of hemostasis. Thrombomodulin and a newly identified receptor for protein C/activated protein C, EPCR, are both present on endothelium. EPCR augments activation of protein C by the thrombin-thrombomodulin complex.
Methods and Results To gain a better understanding of the relationship between thrombomodulin and EPCR, we compared the cellular specificity and tissue distributions of these two receptors by using immunohistochemistry. EPCR expression was detected almost exclusively on endothelium in human and baboon tissues. In most organs, EPCR was expressed relatively intensely on the endothelium of all arteries and veins, most arterioles, and some postcapillary venules. EPCR staining was usually negative on capillary endothelial cells. In contrast, thrombomodulin was detected at high concentrations in both large vessels and capillary endothelium. Both thrombomodulin and EPCR were expressed poorly on brain capillaries. The liver sinusoids were the only capillaries in which EPCR was expressed at moderate levels and thrombomodulin was low. EPCR and thrombomodulin were both expressed on the endothelium of vasa recta in the renal medulla, the lymph node subcapsular and medullary sinuses, and some capillaries within the adrenal gland. Even in these organs the majority of capillaries were EPCR negative or stained weakly.
Conclusions These studies suggest that EPCR may be important in enhancing protein C activation on large vessels. The presence of high levels of EPCR on arterial vessels may help explain why partial protein C deficiency is a weak risk factor for arterial thrombosis.
Key Words: thrombomodulin • vessels • anticoagulants • thrombin • coagulation
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Introduction |
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Protein C is a critical negative regulatory protein of the coagulation cascade, as evidenced by the fact that total deficiencies of protein C lead to life-threatening thrombotic complications in neonates that can be corrected by protein C supplementation1 (also reviewed in Reference 22 ). The protein C zymogen is activated to the anticoagulant APC by a complex between thrombin and the endothelial cell receptor thrombomodulin.3 (For reviews of the overall pathway, see References 4 through 64 5 6 .) In addition to promoting thrombin activation of protein C, thrombomodulin blocks thrombin-dependent fibrinogen clotting and platelet activation. APC functions as an anticoagulant in plasma by inactivating factors Va and VIIIa on membrane surfaces, a process that is potentiated by the plasma vitamin K–dependent factor, protein S.7 8 The importance of factor Va inactivation is illustrated by the clinical observation that the most common form of familial thrombophilia is caused by a polymorphism at residue 506,9 10 11 12 one of the cleavage sites involved in factor Va inactivation by APC.13
With the recent identification14 15 and
cloning15 of the EPCR, this pathway has become more
complex than previously appreciated. EPCR is a type 1 transmembrane
protein16 that is constitutively expressed on cultured
human umbilical cord endothelium and bovine aortic
endothelium.15 Preliminary surveys of
cultured cell lines indicated that EPCR is expressed at high levels
only on endothelium.15 EPCR binds
specifically to either protein C or APC.15 Binding of
protein C to EPCR promotes protein C activation17 and
blocks APC anticoagulant activity. It is unlikely that the
physiological function of EPCR is to inhibit APC
anticoagulant activity, but it may reflect a general change in enzyme
specificity toward a new, as yet unidentified substrate. This
possibility is supported by the observation that inhibition of APC
anticoagulant activity is not due to masking the active site of APC
because the EPCR-APC complex reacts normally with the macromolecular
proteinase inhibitors 
1-antitrypsin and
protein C inhibitor.18 Blocking access of
normal substrates would prevent substrates from competing with the
putative new substrate. This situation is reminiscent of the change in
specificity of thrombin that accompanies thrombomodulin binding. In
this case, the clot-promoting activities of thrombin are blocked,
whereas protein C activation is favored.4
The vascular location of thrombomodulin and EPCR has important ramifications in terms of the mechanisms of protein C activation. In the microvasculature, the surface area of endothelium exposed to blood is much greater than on large vessels, and hence the same thrombomodulin density per cell results in more than a 100-fold increase in the thrombomodulin/blood ratio.3 4 This predicts that protein C activation should occur primarily in the microcirculation and opens the question of whether mechanisms may exist to promote protein C activation selectively within in the larger vessels.
Given a prominent role of EPCR in protein C activation and the demonstrated ability to modulate APC function, we believed that it was important to analyze the cellular specificity and tissue distribution of this newly identified member of the protein C anticoagulant pathway, with particular emphasis on the comparison with the distribution of thrombomodulin. In this article we demonstrate that EPCR expression appears to be quite endothelial cell specific, and that unlike thrombomodulin, the expression of EPCR is largely restricted to veins and arteries, with little expression in the capillaries of most organs.
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Methods |
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Materials
Baboon tissues were obtained from two animals after being given lethal infusion of sodium pentobarbital, after which organs were harvested immediately. The baboons (Papio c anubis) were from a breeding colony maintained at the University of Oklahoma Health Sciences Center. The animals were healthy adults with peripheral blood leukocyte counts of 6000 and 7000/µL, respectively.
Human tissues were collected from nonpathological portions of surgical and autopsy specimens from the University of Oklahoma Health Sciences Medical Center. Surgical tissue samples were obtained within 2 hours of surgical removal. The autopsy tissues were obtained within 12 hours of death: case 1 from a 62-year-old man with acute anterior wall myocardial infarction (6 hour duration), case 2 from a 1-month-old infant with bronchopulmonary dysplasia, and case 3 from a 36-year-old woman with acute pancreatitis. Human biopsy specimens of various tissues (kidney, liver, heart, stomach, skin, lymph node, and striated muscle) that did not show pathological changes in routine histological examination were also selected from the surgical pathology files (paraffin-embedded archival material).
For cryostat sectioning, tissue samples (approximately 6x6x3 mm) were immersed in OCT compound (Miles, Inc) in cryomolds, snap-frozen in liquid nitrogen, and stored at -70°C. For paraffin embedding, the baboon tissues were fixed in 4% phosphate-buffered paraformaldehyde for 18 hours, and the human tissues were fixed in 10% phosphate-buffered formaldehyde for 2 to 12 hours. Specimens submitted for cryostat sectioning and paraffin embedding included representative portions of all of the organs reported in this study.
Antibodies
Three murine mAbs reactive with human and baboon EPCR (1462, an
IgG2Bk; 1489 and 1495, both IgG1k) were prepared
by immunization with a recombinant soluble form of EPCR essentially as
described17 and isolated on a HiTrap Protein G column
(Pharmacia Biotech) from ascites according to the manufacturers
directions. Antibodies 1462 and 1489 stain EPCR in paraffin-embedded
tissues. The working concentrations of 1489, 1495, and 1462 mAbs were
4.5, 9, and 1.5 µg/mL, respectively.
For detection of thrombomodulin expression, a murine mAb (1009, an IgG1k)17 and a goat polyclonal antibody prepared against recombinant soluble human thrombomodulin19 were used. The polyclonal antibody reacts with thrombomodulin in paraffin-embedded human tissues. The working concentration of the 1009 mAb was 15 µg/mL.
For detecting protein C associated with the blood vessel, the murine mAb to human protein C (C8, an IgG1k) was chosen because it cross-reacts with baboon protein C and can be used on frozen sections or paraffin-embedded tissues. The working concentration of the C8 mAb was 1.7 µg/mL.
Immunohistochemistry
Immunohistochemical stainings for EPCR were performed on freshly
prepared cryostat sections (5 µm) and also on paraffin sections
(4 µm) of human and baboon tissues. Cryostat sections were fixed
for 10 minutes in cold acetone (-20°C) and air dried. Paraffin
sections were deparaffinized and rehydrated. All subsequent incubations
and rinses were performed at room temperature. Optimal conditions for
stainings were determined in preliminary experiments. All stainings on
each frozen and paraffin material were performed in duplicate using mAb
1489 and 1495 for frozen material and 1489 and 1462 for the paraffin
material. At least three specimens (from different individuals) from
each of the human tissues studied were examined for EPCR expression
and, except where indicated, gave similar staining patterns.
Before primary antibody incubation, endogenous peroxidase activity was blocked with 1.25% hydrogen peroxide in methanol for 30 minutes. For antigen retrieval, the microwave pretreatment method suggested by Shi et al20 was used with modifications for all EPCR antibodies. Briefly, paraffin sections in 10 mmol/L citric acid buffer (pH 6.0) were heated in a microwave oven for 5 minutes with a 700-W oven set at 50% power. The slides were allowed to cool for 20 minutes at room temperature in the buffer. After preincubation with 10% normal horse serum or 10% normal rabbit serum for 20 minutes, sections were incubated with primary antibodies for 60 minutes, followed sequentially with biotinylated horse anti-mouse (Vector Laboratories) or biotinylated rabbit anti-goat (Dako) antibodies for 20 minutes, and streptavidin-biotin peroxidase complex (Vector Laboratories) for 20 minutes. The reaction was developed with diamino-benzidine (Sigma Chemical Co), and the sections were counterstained with Mayer's hematoxylin. All antibodies were diluted in phosphate-buffered saline (PBS, pH 7.4) containing 1% bovine serum albumin. Between the incubation steps, the slides were washed twice in PBS for 10 minutes.
Thrombomodulin expression was demonstrated on cryostat (5 µm) and paraffin (4 µm) sections of human tissues. The immunohistochemical staining for thrombomodulin was similar to that described above for EPCR except that (1) the sections were not treated in the microwave, (2) the blocking serum was either 10% normal horse (for antibody 1009) or rabbit serum (for polyclonal goat anti-human thrombomodulin antibody), and (3) the secondary antibody was either biotinylated horse anti-mouse (for antibody 1009) or biotinylated rabbit anti-goat (for polyclonal goat anti-human thrombomodulin antibody).
Protein C staining was performed on paraffin-embedded human lung and various baboon tissues. The staining procedure was similar to that described above for the mAbs against EPCR, which included microwave treatment for antigen retrieval. The tissues were fixed before paraffin embedding in an effort to minimize protein C dissociation in subsequent steps of tissue processing and staining.
As a negative control, stainings on sequential sections of each tissue were performed with substitution of the primary antibody with either mouse monoclonal IgG1 standard (Bethyl Labs) or preimmune goat serum with appropriate dilution. The nonspecific staining was negligible.
Evaluation of the immunohistochemical stains was conducted in a semiquantitative manner. All stainings were evaluated as signal/noise (image/background) intensity. Scoring of the staining intensity was given a scale of (-) to 4+. The positive range of scores was assessed as follows: 1+ (weak, but well recognizable staining); 2+ (moderate staining); 3+ (strong staining); and 4+ (very strong staining).
Evaluation of the distribution of EPCR from various human and baboon tissues with three EPCR mAbs with different epitopes yielded identical results, indicating that the staining was due to the presence of EPCR and not to cross-reacting material. No differences were noted in the staining patterns between frozen and paraffin material.
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Results |
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To gain an initial appreciation of the potential interactions of EPCR and thrombomodulin in the regulation of the protein C anticoagulant pathway, human tissues were evaluated for EPCR (Fig 1
, left) and thrombomodulin (Fig 1, right)
expression. For purposes of comparison, to avoid potential disease
influences on EPCR expression, and to minimize the potential for post
mortem changes in EPCR expression, we also examined the EPCR
distribution and vascular location in baboon tissues (Fig 2). With few exceptions, the EPCR
distribution was the same in baboon and human autopsy and surgical
tissues, and the small differences are discussed under the description
of individual organs. For clarity of discussion, the pictures in Fig 1
and Fig 2 are of human and baboon tissues, respectively. At least three
(usually more) representative sections were examined
for each organ. These were analyzed at both high and low
magnifications. The photographs in Figs 2 through 4 are
representative in all tissues, EPCR expression was
essentially endothelial cell specific. Lung and heart
were representative of most tissues. EPCR was expressed
most strongly and consistently in large arteries, with the
large veins expressing similar or perhaps slightly lower levels of EPCR
(Fig 1 (left), A [heart] and B [lung]). Postcapillary venule
staining was variable, ranging from negative to relatively intense
(see below). In contrast, most capillary endothelial
cells were EPCR negative in heart and lung.
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EPCR Evaluations
Heart. In the heart, in addition to the staining
described above, EPCR staining was strongly positive (3+) on the
endocardium including both ventricles, atria, appendages, and valves
(Fig 2A
). The staining of some postcapillary venules can be seen in Fig 2A and more clearly at higher magnification (Fig 2B). At higher
magnification, the intense staining of all of the
endothelial cells in a large coronary artery
(Fig 2C) is in stark contrast to the generally negative staining of the
capillaries as seen in Fig 2B. Similar EPCR staining to that seen in
baboons was observed in human surgical and autopsy tissues, suggesting
that post mortem changes, tissue harvesting, and possible disease
processes in the humans probably did not dramatically influence the
expression of EPCR. The capillary endothelial cells
were usually negative, with 
5% scattered
endothelial cells (referred to as "patchy" in the
Table) staining weakly for EPCR in both
baboon and human heart tissue. One exception was the capillary
endothelium from the infant with
bronchopulmonary dysplasia (autopsy case 2), in which weak to
moderate (1 to 2+) EPCR staining was observed in 50% of the
capillaries (data not shown). Although no histopathological changes in
the myocardium were apparent in this case, the possibility
that the widespread EPCR staining of the capillaries was due to the
disease process or young age of the individual cannot be excluded.
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As opposed to EPCR, thrombomodulin was widely expressed in the
endothelial cells of the human heart including the
coronary arteries, veins, postcapillary venules, and
capillaries (Fig 1A, right).
Lung. The arteries, including the small intraacinar and
intra-alveolar arteries, and veins were strongly EPCR positive (3+)
(Fig 1B, left). There were very few (
1%) scattered
endothelial cells in the alveolar walls that were EPCR
positive. In contrast, the alveolar endothelial cells
stained intensely for thrombomodulin (Fig 1B, right). The staining
pattern for EPCR was similar in baboons and humans.
Skin. The dermal arteries, arterioles, and veins were
strongly (3+) EPCR positive; some of the dermal capillaries revealed
weak to moderate (1 to 2+) staining (Fig 1C, left). The epidermis was
negative with only a slight (1+) staining of the intercellular
bridges. In contrast, as reported previously,21
thrombomodulin expression was observed not only in all of the
endothelial cells of the skin but also in the squamous
epithelium (Fig 1C, right). Thus in the skin, EPCR is more
endothelial cell specific than thrombomodulin, as well
as being more restricted to larger vessels. The baboon and human skin
had a similar EPCR staining pattern.
Liver. Endothelial cells of the liver
revealed strong (3+) EPCR staining in the central veins, portal vein,
and hepatic arteries and moderate (2+) staining in the sinusoidal
capillaries (Fig 1D, left). The staining of these sinusoidal
capillaries for EPCR was more intense than in the capillaries of other
tissues examined. In contrast, thrombomodulin stained intensely
in the larger vessels within the liver, but thrombomodulin
staining of the sinusoidal capillary endothelium was
less intense (negative to 1+) than EPCR (2+) (compare Fig 1D, left and
right). The staining pattern for EPCR was similar for human and baboon
liver.
Brain. Strong (3+) EPCR expression was detected in the
arteries and veins of the subarachnoid space of the brain (Fig 2D). Many of the arteries, veins, and postcapillary venules of the
white matter but only some in the gray matter were moderately to
strongly (2 to 3+) positive; the majority of capillaries in the white
and gray matter were negative, with the others staining weakly (1+) for
EPCR. In addition, all of the epithelial and most of the
endothelial cells of the choroid plexus of the fourth
ventricle showed 1+ and 2+ staining, respectively. Staining patterns
for human and baboon brain were similar. We confirmed previous
observations22 that thrombomodulin is expressed at high
levels on large vessels in the human brain and at low levels, similar
to EPCR, in the brain capillaries (data not shown).
Kidney. Moderate to strong EPCR staining (2 to 3+) was
seen in the interlobular and arcuate arteries, veins, and in
some of the arterioles. The glomerular capillary
endothelial cells and the great majority of the
cortical tubulointerstitial capillary
endothelial cells were negative (Fig 2E). The few
cortical tubulointerstitial capillary
endothelial cells that were EPCR positive were most
conspicuous around the tubules in close vicinity to large veins. The
medullary vasa recta and many of the
tubulointerstitial capillary
endothelial cells at the
corticomedullary junction stained positively (2+)
in both humans and baboons (Fig 2F). In baboons, there were occasional
glomeruli, with a few weakly positive (1+) glomerular
capillary endothelial cells. Although not visible in
Fig 2E, these positive glomerular
endothelial cells were located primarily at the
glomerular stalk. In human kidneys with no associated
histopathological changes, the cortical
tubulointerstitial capillary staining for EPCR was
even more sparse and weaker than in baboons (data not shown).
Adrenal. Weak to moderate staining of EPCR (1 to 2+) was
detected in the endothelial cells of the zona
reticularis and zona fasciculata in baboon (Fig 2G) and human adrenal
cortex. In addition, there was moderate EPCR staining in the small
veins and postcapillary venules of the adrenal medulla.
Uterus. The arteries, veins, and many of the venules in the
myometrium were strongly EPCR positive. There was strong (3+) EPCR
staining in the spiral arteries of the endometrium and weak (1+)
staining in some of the endometrial capillaries (Fig 2H). The staining
pattern was similar in baboons and humans.
Lymph nodes. There was strong (3+) EPCR staining in the
subcapsular and medullary sinuses in both baboons and humans (Fig 3A
and B). For comparison in Fig 3A, the staining of a small artery is
only slightly more intense than subcapsular sinusoidal
endothelial cells. The high endothelial
venules were negative.
Spleen. The trabecular veins and arteries and
the follicular arterioles were strongly EPCR positive (3+). The venous
sinusoids of the red pulp were either negative or slightly focally
positive in the baboon and in two of the three human autopsy specimens
(1+). In one of the human autopsy cases (case 3), the
endothelial lining of the venous sinusoids of the red
pulp was diffusely moderately positive (2+) (Fig 4A).
Aorta, large muscular arteries, and veins. A very strong
staining (4+) was limited to the endothelial cells in
these vessels (Fig 4B). The EPCR distribution in other tissues is
summarized in the Table.
Protein C binding to endothelium. EPCR was
originally identified on the basis of protein C binding. If EPCR served
as a protein C binding protein in vivo, then the vessels expressing the
highest levels of EPCR should also bind protein C most intensely.
Consistent with this possibility, protein C immunoreactivity
(Fig 5A) in baboons and humans mirrored
that of EPCR (Fig 5B) with protein C immunoreactivity detected on
arteries, veins, and venules of various organs and negative in
capillaries (Fig 5A). The lone exception was the endocardium, in which
EPCR was strongly positive but protein C was consistently
negative. The basis for the discrepancy is not known. It is possible
that EPCR in the endocardium exists in an inactive form on the cell
surface or is largely intracellular, that protein C dissociated during
processing or, less likely, that EPCR is not a major protein C–binding
protein.
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Discussion |
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The current study demonstrates that EPCR expression in vivo is restricted primarily to the endothelium. In contrast to thrombomodulin, which is abundant both in large vessels and most capillaries, in most organs EPCR expression is restricted primarily to veins and arteries, with most capillary endothelial cells expressing little if any EPCR. Capillary expression of EPCR is detectable, however, in some specialized capillary beds. For example, medullary capillaries in the kidney and the subcapsular and medullary sinuses of the lymph nodes stained positively for EPCR. Liver sinusoidal endothelium is a rare exception in that EPCR expression is positive and thrombomodulin negative to weak. Expression within postcapillary venules varies from moderate to undetectable within the same region of the same organ. The arteries consistently showed somewhat higher levels of expression than veins of similar size. As a general observation, EPCR staining intensity increases with increasing vessel size.
From our current understanding of protein C activation, the differences
in distribution between thrombomodulin and EPCR suggest major
differences in the properties of the protein C activation complex on
large vessels compared with capillaries. First consider the effective
thrombomodulin concentrations in these two vessel types. Because of
geometric considerations (endothelial cell surface to
blood volume ratios) discussed previously4 23 and with the
assumption that there are 50 000 thrombomodulin molecules per
endothelial cell24 on both large and small
vessels, then the effective thrombomodulin concentration in the large
vessels would be >100-fold lower than in the capillaries (100 to
500 nmol/L). By augmenting protein C activation,17
EPCR may help to compensate for the relatively low concentration of
thrombomodulin in large vessels. Second, since the Km of
the activation complex for protein C is reduced by EPCR,17
the activation complexes on the arteries containing EPCR would be
assumed to be less sensitive to changes in protein C concentration than
the activation complexes within the capillaries having little or no
EPCR. These properties of the activation complex may help explain the
observation that heterozygous protein C deficiency is at most a weak
risk factor for arterial thrombosis.25 The
vascular distribution and properties of EPCR lead to the prediction
that deficiencies of EPCR might constitute a risk factor for
arterial thrombosis, especially if concomitant protein C
deficiency were present.
One obvious question to arise from these studies is why EPCR expression is so low within most capillaries. Clearly the answer is not known, but some predictions can be made about the biological importance of this observation in terms of known functions for EPCR and thrombomodulin. For instance, thrombomodulin has at least two functions: to augment the thrombin-dependent activation of protein C and to enhance the reactivity of thrombin with antithrombin26 and the protein C inhibitor.27 When EPCR is present, each thrombin-thrombomodulin complex is a more efficient protein C activator. Thus EPCR enhances protein C activation without any known influence on thrombin clearance. In the capillaries, protein C activation is presumably less efficient because of the paucity of EPCR. Thus each thrombin-thrombomodulin complex activates less protein C before being inactivated by antithrombin or protein C inhibitor. This would result in a net shift in thrombomodulin function toward thrombin clearance. The high thrombomodulin concentration in the microcirculation allows for high thrombin binding capacity, potentially allowing this mechanism to play a major role in thrombin clearance.
Protein C and APC have been implicated as important factors in blocking tissue injury in gram negative sepsis. In experimental animals, the evidence in favor of this concept is that APC can protect rodents28 and primates29 from lethal levels of Escherichia coli, and blocking the protein C pathway exacerbates the response to low-level bacterial infusion and elevates the cytokine response.29 30 Clinically, the extent of protein C consumption correlates well with negative clinical outcomes in meningococcemia.31 Preliminary clinical results have suggested that replacement therapy with protein C results in blocking the progression of the disease process in most patients32 33 and a rapid improvement in organ function. For these reasons, we had considered EPCR a candidate for modulating the inflammatory response and had hypothesized that EPCR might be colocalized with key leukocyte adhesion receptors. Many of the receptors involved in leukocyte–endothelial cell interaction are located in the postcapillary venules, where leukocyte trafficking occurs,34 and not normally on large vessels. The presence of EPCR on some but not all postcapillary venules would be consistent with this potential function in at least some vascular beds. Whether EPCR can be induced in other postcapillary venules in response to inflammatory stimuli is currently under investigation. Consistent with the possibility, preliminary analysis at the whole organ level suggests that EPCR message rises as an early immediate response in rodents challenged with endotoxin.35 Inflammatory mediators also may be responsible for some of the relatively minor differences in EPCR expression, especially in capillaries, noted in some of the tissues. Alternatively, the possibility that these differences are related to different species cannot be excluded.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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These studies were supported by grants awarded from the National Institutes of Health, Grant Nos. PO1-HL-54804 and R37-HL-30340 (to C.T.E.) and R01-GM37704 (to F.B.T.). Dr Esmon is an investigator of the Howard Hughes Medical Institute. The authors would like to thank Drs Naomi Esmon, Debbie Stearns-Kurosawa, and Shinichiro Kurosawa for their helpful suggestions and Julie Wiseman for help in preparation of the final manuscript.
Received April 24, 1997; revision received July 9, 1997; accepted July 15, 1997.
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