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Endothelial Cell Protein C Receptor
Role Beyond Endothelium?
Meenakshisundaram Thiyagarajan, Tong Cheng, Berislav V. Zlokovic
E
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ndothelial cell protein C receptor (EPCR) is a type 1
transmembrane glycoprotein with homology to the
major histocompatibility complex (MHC)-class
1/CD1 family of molecules.1 EPCR was isolated and cloned
as an endothelial cell-specific, high selectivity and high
affinity binding protein for protein C (PC) and activated PC
(APC).1 EPCR binds PC on the endothelial surface and
presents it to the thrombin:thrombomodulin (TM) complex
for activation (Figure 1). Thrombin bound to TM proteolytically activates PC and generates APC which exerts independent anticoagulant and cellular activities.2 The APC anticoagulant pathway is mediated by proteolytic inactivation of
blood coagulation Factors Va and VIIIa with the contribution
of several cofactors, including protein S, high density lipoproteins, phosphatidylserine, cardiolipin, and glucosylceramide to name a few. The APC cellular pathway on endothelium requires EPCR and the protease activated receptor-1
(PAR-1)3 which mediates APC’s cytoprotective effects, including alterations in gene expression profiles, antiapoptotic
activity, antiinflammatory activity and protection of endothelial barriers4 –10 (Figure 1). EPCR also regulates endogenous
physiologic activation of PC by thrombin which is linked to
PAR-1-dependent APC protective autocrine signaling in
endothelium.11
EPCR signaling can decrease inflammation. APC binding
to EPCR rescues baboons from E. coli sepsis.12 EPCR has
also cardioprotective role in lipopolyscharide-induced endotoxemia in mice.14 In addition to the cell-surface EPCR,
soluble EPCR lacking the transmembrane helix of native
EPCR interacts with the integrin CD11b/CD18 (Mac-1)
(␣M␤2) (CR3) on leukocytes (Figure 1), suggesting that
binding of soluble EPCR to Mac-1 might regulate leukocytes
adhesion.15 Proteinase-3 (PR3), a serine protease with elastase-like properties stored in granules of neutrophils, binds
both Mac-1 and soluble EPCR which may be implicated in
APC mediated signaling and activation of PC on leukocytes,
because soluble EPCR:PR3 complexes bind both APC and
PC.15
Recent evidence indicates that EPCR is expressed on
different cells beyond aortic endothelial cells. For example,
EPCR is expressed on the surface of monocytes, CD56⫹
natural killer cells, neutrophils and eosinophils16 –18, immature hematopoietic stem cells,19 brain capillary endothelial
cells.13,20 EPCR is also expressed by embryonic giant trophoblast cells, and EPCR is critical for embryo development,
because EPCR null mice die in mid-gestation.21 The study by
Bretschneider et al, in the present issue of Circulation
Research demonstrates functionally active EPCR in systemic
vascular smooth muscle cells (SMCs).22 The data show that
EPCR mediates APC signaling in SMCs, resulting in increases in intracellular [Ca2⫹]i, phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK-1/2) and DNA
synthesis (Figure 1). Along with EPCR, PAR-1 is also
necessary to transduce APC signaling in SMCs.
These findings are consistent with previous work demonstrating that both EPCR and PAR-1 are required for APCmediated activation of ERK1/2 and [Ca2⫹]i signaling in
systemic and cerebral capillary endothelial cells.5,20 These
findings open a new chapter in biology of EPCR by demonstrating that EPCR can be expressed in cells which have
different biological functions from endothelial and hematopoetic cells. In this context, it is of note that the microarray
gene expression profiling of human cerebral SMCs and
astrocytes have indicated the presence of EPCR mRNA and
showed that the levels of EPCR transcripts in these brain cells
were not altered by normal aging in humans or aging
Alzheimer’s type (R. Zidovetski, N. Chow, B. Zlokovic,
unpublished data, 2007). The exact role of EPCR and how its
expression is regulated in nonendothelial cell types including
systemic SMCs, primitive hematopoetic stem cells or cerebral
SMCs, remain to be elucidated.
A regulatory element 5.5 kb upstream to the human EPCR
gene (hEPCR) translation start site is essential for driving
hEPCR expression in endothelium and primitive hematopoetic cells which share a common precursor.23 It would be
interesting to find out whether the same site in hEPCR is
responsible for driving hEPCR expression in human SMCs.22
The present findings by Bretschneider et al22 raise not only a
possibility that EPCR could play an important role in regulating the biology of vascular SMCs, but also suggest that its
expression in SMCs might have implications for the pathogenesis of cardiovascular diseases such as atherosclerosis,
and by extension, cerebral amyloid angiopathy in Alzheimer’s disease. EPCR may be a potential therapeutic target:
novel EPCR agonists might modify cytoprotective APC/PC
cellular pathways in injured or inflamed blood vessels.
We still have much to learn about the role of EPCR beyond
the endothelium, and perhaps in the endothelium itself. For
The opinions expressed in this editorial are not necessarily those of the
editors or of the American Heart Association.
From the Frank P. Smith Laboratory for Neuroscience and Neurosurgical Research (M.T., T.C., B.V.Z.), Department of Neurosurgery,
University of Rochester Medical Center, NY.
Correspondence to Berislav V. Zlokovic, MD, PhD, University of
Rochester Medical Center, 601 Elmwood Avenue, Box 645, Rochester,
New York 14642. E-mail [email protected]
(Circ Res. 2007;100:155-157.)
© 2007 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/01.RES.0000258167.48227.84
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Circulation Research
February 2, 2007
EPCR
Leukocyte
PAR-1
sEPCR
Mac1
Va & VIIIa
Vi & VIIIi
PR3
Inflammatory
Mediators
APC
Thrombin
Y
PC
APC
EPCR
PAR-1
PLC IP3
EPCR
TM
Gi Gq
NF-kB
Pro-Inflammatory
genes
2+
p53
Ca
Bcl-2
Bax
2+
Ca regulated
Kinases
Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017
Apoptosis
activation of
initiator caspases
Mitochondria
ERK1/2
Nucleus
pERK1/2
DNA Synthesis
Death
Receptors
example, Dr Esmon has suggested that EPCR appears to be
able to translocate from the plasma membrane into the
nucleus, a cellular trafficking pattern that is not observed with
its Cys to Ser mutant.24 One may speculate that EPCR may
internalize its endogenous ligands PC and APC into the
cytoplasm and into the nucleus, which in turn could have
important implications for regulation of gene expression and
control of APC activity. Figure 2a shows dense punctate
staining of APC on the surface of brain endothelial normoxic
cells, which is completely removed by pretreatment of cells
with an antibody that blocks APC binding site on EPCR
(Figure 2b),25 demonstrating that APC binding to the cell is
highly EPCR-dependent, as suggested.1 On the other hand,
A
B
Figure 1. EPCR binds protein C (PC) on endothelium and presents it to TM
(thrombomodulin)-thrombin complex for activation by thrombin. Activated PC (APC) dissociates from EPCR and TM-thrombin complexes and inactivates coagulation factors Va
and VIIIa into inactive forms Vi and VIIIi promoting anticoagulation. APC bound to EPCR
activates the protease activated receptor-1
(PAR-1) which binds the Gq and Gi proteins to
activate phosophlipase(s) (PLC) in SMCs and
endothelium resulting in IP3 (inositol
triphosphate)-mediated release of intracellular
[Ca2⫹]i, activation of mitogen-activated
kinases ERK1/2 and stimulation of DNA synthesis.22 In injured endothelium, EPCR-APCmediated PAR-1 activation blocks
mitochondria-mediated apoptotic pathway
(eg, inhibition of p53 and Bax, activation of
antiapoptotic Bcl-2 gene, blockade of
caspase-9), and death receptor-mediated
pathway through inhibition of caspase-8.
Whether the same antiapoptotic signaling
exists in injured SMCs is unknown. Soluble
form of EPCR (sEPCR) binds APC/PC and
forms complexes with proteinase-3 (PR3) which
binds to Mac-1 on leukocytes and activates
antiinflammatory signaling. EPCR-APC– dependent PAR-1 activation in leukocytes and endothelium blocks the expression of proinflammatory genes by preventing nuclear translocation
of nuclear factor-␬B (NF-␬B). EPCR translocates
itself from the plasma membrane to the nucleus
and may internalize APC into injured vascular
cells, but the physiological importance of EPCR
internalization alone and/or of its ligands
remains at present elusive.
Figure 2c shows that APC can be transported intracellularly
and into the nucleus of brain endothelial cells under hypoxic
conditions (Figure 2c), which is associated with cell protection and survival, as reported.13 This transport pattern, however, completely disappears if APC binding to EPCR is
blocked (Figure 2d), and cells die as a result of APC
unavailability, as shown by faint APC staining on cell debris.
Thus, the role of EPCR-APC-PAR-1-dependent intracellular
cytoprotective signaling and of EPCR-mediated intracellular
trafficking and internalization of APC/PC in normal and
injured vascular endothelial cells and SMCs, may open a new
era in our understanding of EPCR biology, its physiological
and pathological functions, and therapeutic opportunities for
C
D
Figure 2. Human brain endothelial cells were cultured under normoxic and hypoxic (⬍ 1% oxygen, no glucose) conditions, as we
described.10,13,20 Human plasma-derived APC (20 nM) was incubated with cells for 4 hour under either normoxic (A and B) or hypoxic
(C and D) conditions in the absence (A and C) and presence (B and D) of an anti-EPCR antibody (RCR-252) which blocks APC binding
site on EPCR.25 APC binding was detected by an APC/PC-specific antibody (C3), as described,11,13 using confocal microscopy. In normoxic cells, a punctate staining shows binding of APC to the plasma membrane (A) and complete displacement of APC binding by
anti-EPCR antibody (B). In hypoxic cells, APC is internalized into the cytoplasm and nucleus which is associated with the cell survival
(C). In contrast, anti-EPCR antibody prevents APC binding to hypoxic cells which is associated with loss of protection and cell death
(D), as reported.13
Thiyagarajan et al
disorders of the cardiovascular system and cerebral
circulation.
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KEY WORDS: endothelial protein C receptor 䡲 activated protein C
䡲 protease activated receptor-1 䡲 endothelium 䡲 vascular smooth muscle cells
Endothelial Cell Protein C Receptor: Role Beyond Endothelium?
Meenakshisundaram Thiyagarajan, Tong Cheng and Berislav V. Zlokovic
Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017
Circ Res. 2007;100:155-157
doi: 10.1161/01.RES.0000258167.48227.84
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