Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:178-182
(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:178.)
© 2001 American Heart Association, Inc.
Thromboregulation by Endothelial Cells
Significance for Occlusive Vascular Diseases
Aaron J. Marcus;
M. Johan Broekman;
Joan H.F. Drosopoulos;
David J. Pinsky;
Naziba Islam;
Richard B. Gayle, III;
Charles R. Maliszewski
From the VA New York Harbor Healthcare System and Weill Medical College
of Cornell University (A.J.M., M.J.B., J.H.F.D., N.I.), New York, NY; Columbia
University College of Physicians and Surgeons (D.J.P.), New York, NY; and
Immunex Corp (R.B.G., C.R.M.), Seattle, Wash.
Correspondence to Aaron J. Marcus, MD, Chief, Hematology/Medical Oncology, VA New York Harbor Healthcare System, 423 East 23rd St, Room 13028W, New York, NY 10010. E-mail ajmarcus{at}med.cornell.edu
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Abstract
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AbstractDuring
their 7- to 9-day lifespan in the circulation,
platelets perform an
ill-defined baseline function that maintains
the integrity of the
vasculature. In thrombocytopenic states,
there is an increase in
vascular permeability and fragility,
which is presumably due to absence
of this platelet function.
In sharp contrast, biochemical or
physical injury in the coronary,
carotid, or
peripheral arteries induces platelet activation
and
platelet recruitment, which can progress to thrombotic vascular
occlusion.
Because there is 1 death every 33 seconds from vascular
occlusion
in the United States, this problem has critical public health
implications.
In this review, we describe the characterization of a
novel
potential antithrombotic agent with a unique mode of
actionbiochemical
"deletion" of ADP from an activated platelet
releasate, which
thereby inhibits platelet recruitment and further
activation.
Key Words: endothelial cells thrombosis platelets CD39/ecto-ADPase
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Introduction
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Several
years ago, in the course of studying interactions between
cells of the
vascular wall and those in the circulation, we
demonstrated that
platelets in proximity to endothelial cells
do not
respond to any added agonist in vitro. Experiments initiated
in the
late 1980s cumulatively led to the conclusion that
endothelial
cell CD39, an ecto-ADPase, was mainly
responsible for this phenomenon.
CD39 rapidly and preferentially
metabolizes ADP released from
activated platelets. ADP is
the final common pathway for platelet
recruitment and thrombus
formation, and platelet aggregation
and recruitment are abolished
by CD39. Our present working hypothesis
is that CD39
represents a novel antithrombotic agent for treating
high-risk
patients who have activated platelets in their circulation,
the
identifying characteristic of coronary artery occlusion and
thrombotic
stroke.
We generated a recombinant soluble form of human CD39
(solCD39), a glycosylated protein of 66 kDa whose enzymatic and
biological properties are identical to the full-length form of this
enzyme. In our in vitro experiments, solCD39 blocks ADP-induced human
platelet aggregation and inhibits collagen-induced and thrombin
receptor agonist peptide (TRAP)-induced platelet reactivity. We
also studied solCD39 in vivo in a murine model of stroke, which was
shown to be driven by excessive platelet recruitment. In studies
with CD39 wild-type (CD39+/+) mice, solCD39
completely abolished ADP-induced platelet aggregation and strongly
inhibited collagen- and arachidonate-induced platelet
reactivity ex vivo. When solCD39 was administered before transient
intraluminal middle cerebral artery occlusion, it reduced ipsilateral
fibrin deposition, decreased
111In-platelet deposition, and increased
postischemic blood flow 2-fold at 24 hours. These results
were superior to those we obtained with aspirin
pretreatment.
CD39 null (CD39-/-) mice, which
we generated by the deletion of exons 4 to 6 (apyrase-conserved regions
[ACRs] 2 to 4), have a normal phenotype and normal
hematologic profiles and bleeding times, but they exhibit a decrease in
postischemic perfusion and an increase in cerebral infarct
volume when they are compared with genotypic
CD39+/+ control mice in our stroke model.
"Reconstitution" of CD39 null mice with solCD39 reversed these
pathological changes. Thus, the CD39-/- mice
were actually rescued from cerebral injury by solCD39, thereby
fulfilling Kochs
postulates.1 These experiments
have led us to hypothesize that solCD39 has potential as a novel
therapeutic agent for thrombotic stroke.
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Interactions Between Platelets and
Vascular Endothelium
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Thrombosis is a multicellular
process.
2 To gain further
insight
into the pathogenesis of vascular occlusion, cell-cell
interactions
must be studied in detail. Cell-cell interactions and
interactions
between blood cells and the vessel wall are critical
components
of the hemostatic and thrombotic process. Several of such
reactions
occur via "transcellular metabolism," a
locution we developed
to indicate reciprocal or collaborative
metabolic steps initiated
by signaling molecules from
different cell types.
3 These
steps
are particularly relevant with regard to vascular
endothelial
cells and platelets. We have
demonstrated that endothelial cells
in proximity to
platelets in motion will downregulate their
activity via at least 3
different metabolic pathways. The first
involves a
cell-associated aspirin-insensitive nucleotidase,
ecto-ADPase/CD39.
4 The second
system consists of a short-lived fluid-phase inhibitory
signaling
pathway involving eicosanoids such as prostacyclin
(PGI
2) and
prostaglandin
D
2.
5
The third is the NO system, which is also
a fluid-phase autacoid that
inhibits platelet reactivity by
elevating levels of
cGMP.
6 We termed these
substances "thromboregulators."
When PGI
2
and NO activities are blocked by aspirin and hemoglobin,
respectively,
platelet inhibition is solely due to the metabolism
of
ADP in the platelet releasate after agonist-induced
platelet
reactivity. Under these conditions, metabolism
of ADP by CD39
results in the loss of platelet responsiveness,
release, recruitment,
and
aggregation.
4 It is important
to emphasize that platelet
inhibition by CD39 is unique because
there is no other consequent
blockade of platelet function per se;
the deletion of ADP from
the activated platelet releasate
is solely responsible for the
abolition of platelet recruitment.
This is the modus operandi
of CD39 as an inhibitor of
platelet aggregation and recruitment.
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Characterization of the Human
Endothelial Cell Ecto-ADPase as CD39
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Our initial studies of the platelet
inhibitory activity of endothelial
cells
during the 1970s and 1980s led to the conclusion that
this property was
due to endothelial cell eicosanoid and/or
NO
production. This early hypothesis was tested in 1990 by
incubating
aspirin-treated human umbilical vein
endothelial cells (HUVECs)
with radiolabeled ADP. Under
these experimental conditions,
PGI
2 could not
form, and any NO that was generated either rapidly
decayed or was
blocked by our addition of purified oxyhemoglobin
to the system. The
added ADP and any metabolites formed were
separated and identified by
radioactive thin-layer chromatography
(Figure
1

). Under these experimental conditions, AMP
accumulated transiently
and was further metabolized to
adenosine, followed by deamination
to inosine and hypoxanthine.
Importantly, these experiments
also demonstrated that cell-free
supernatants from HUVECs incubated
with
[
14C]ADP could not induce aggregation in
platelet-rich
plasma because the ADP had been metabolized by a
component of
the endothelial cell surface, presumably
the ecto-nucleotidase
system.
4

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Figure 1. Metabolism of ADP by endothelial cells (ECs). HUVECs were incubated as indicated with 15 µmol/L [14C]ADP. ADP metabolites were separated by radioactive thin-layer chromatography. The activity of 5'-nucleotidase, as well as adenosine deaminase, is inferred from the rapid appearance of adenosine (Ado) and final accumulation of inosine in these scans. Hypo indicates hypoxanthine.
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We now know that the molecule primarily responsible for
platelet inhibition as described above is indeed an ADPase.
Specifically, it is a membrane-associated ecto-nucleotidase of the E
type.7 8 This enzyme
has several interesting characteristics, which include the following:
it is dependent on calcium (although magnesium can partially
substitute) but not affected by specific inhibitors of P-,
F-, and V-type ATPases, and it metabolizes ATP and ADP but not AMP.
These biochemical activities identify the HUVEC enzyme as an apyrase
(ATP diphosphohydrolase [ATPDase]),
EC3.6.1.5,7 now classified as
E-NTPDase-1.8
In 1996, Handa and
Guidotti9 purified a soluble
apyrase from potato tubers and cloned its cDNA. Sequence
analysis revealed 25% amino acid identity and 48% amino acid
homology with human CD39.9
Maliszewski et al10 had
cloned CD39 as a cell-surface glycoprotein expressed on
activated B cells. It was also present on NK cells and
subsets of T cells as well as on some preparations of
HUVECs.11 Interestingly,
nucleotidases with homology to CD39 and potato apyrase are expressed
throughout the animal and vegetable kingdoms, in species as varied as
the garden pea, Caenorhabditis
elegans, and
Toxoplasma.9
At least 4 regions within these molecules demonstrate extraordinary
homology and are designated
ACRs.9
Several observations have indicated that the HUVEC ADPase is
identical to CD39.12 More
than 95% of the ADPase activity from a preparation purified from HUVEC
membranes can be immunoprecipitated with several anti-human CD39
antibodies. Confocal microscopy and indirect
immunofluorescence studies localized CD39 to the
cell surface of HUVECs. When COS cells are transfected with a vector
containing cDNA from either human or murine CD39, the expression of
CD39 and ecto-ADPase activity are demonstrable immunologically and
biochemically, respectively, on the COS cell surface. Polymerase chain
reaction (PCR) analyses using either authentic human CD39 cDNA
or cDNA synthesized from HUVEC mRNA will result in products of
identical size for each of 4 different CD39-specific primer pairs. When
these PCR products were sequenced, their complete identity was
confirmed. The PCR products encompassed 75% of the coding region
of CD39,12 including the
original 4 ACR apyrase domains
(Figure 2
). Furthermore, Northern analyses
demonstrated that HUVEC and MP-1 cells (from which CD39 was originally
cloned) contain the same sized messages for
CD39.12 Other laboratories
have reported ATPDases from different cell
sources.13 14

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Figure 2. Domain structure of ecto-ADPase/CD39. Two transmembrane regions are located near the amino and carboxy termini; a hydrophobic sequence is centrally located. The putative ACR is shown on the left side as the apyrase domain, adjacent to the N-terminal portion. Cysteine residues are marked as C. An engineered form of soluble CD39, containing a Flag tag and interleukin (IL)-2 secretion leader and lacking the 2 transmembrane regions, is presented below for comparison.
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When COS cells were transfected with human or murine CD39
cDNA, they blocked ADP-induced platelet
aggregation.12 We found that
the transfectants metabolized ADP to AMP within 3 minutes. This time
frame correlates with the events leading to the formation of a
hemostatic platelet plug or thrombus. This event parallels the time
course for platelet inhibition by cells expressing CD39 and is also
commensurate with the respective ADPase activities of these cells
(Figure 3
). The data emphasize the importance of CD39 as a
thromboregulator and represent the first direct demonstration
of a physiological function for CD39 as an
ADPase: blockade of platelet
responsiveness to the prothrombotic agonist ADP via its
metabolism to AMP. This phenomenon might represent
the evolution of an endothelial mechanism targeted
toward metabolism of prothrombotic platelet-derived
nucleotides, thereby controlling excessive platelet
accumulation as well as maintaining blood
fluidity.

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Figure 3. Blockade and reversal of platelet aggregation to ADP by intact HUVECs, MP-1 cells (an activated B-cell line10 ), and COS cells transfected with full-length human or murine CD39. Platelet-rich plasma (PRP) from a donor who had ingested aspirin (ASA) was stimulated with 10 µmol/L ADP in the presence of COS cells transfected with "empty" vector (A), in the absence of any additions (B), and in the presence of MP-1 cells (C), HUVECs (D), and COS cells transfected with human CD39 (huCD39, E) and murine CD39 (muCD39, F). Expression of CD39 led to metabolism of the ADP component of the platelet releasate and acquisition of platelet inhibitory activity due to blockade of platelet recruitment.
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Recombinant Soluble CD39/Ecto-ADPase
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The biochemical and biological properties of CD39
suggest an
entirely new strategy for therapeutic intervention in
platelet-driven
occlusive vascular diseases. Although treatment of
patients
with aspirin inhibits the prothrombotic action of
thromboxane,
it simultaneously prevents the
formation of the antithrombotic
eicosanoid,
PGI
2. This represents a limitation in
the therapeutic
use of aspirin. Moreover, aspirin is a
nondiscriminating acetylating
agent, as we had demonstrated in
1970,
15 and its use is
accompanied
by undesirable side effects. CD39 is aspirin independent
and
inhibits platelet reactivity even when eicosanoid formation
and
NO production are blocked. Importantly, the action of CD39
is
not on the platelet itself; rather, it metabolizes ADP appearing
in
the activated platelet releasate. This action serves to
abolish
platelet recruitment.
Because we found that ADPase/CD39 is an effective
physiological and constitutively expressed
endothelial cell inhibitor of platelet
reactivity, we postulated that a soluble form of the human enzyme would
represent a promising new systemic antithrombotic modality for
thrombosis-prone patients with a low threshold for platelet
activation to be evaluated in vivo and ex vivo. This could be
accomplished in the setting of acute, subacute, or chronic clinical
situations. Thus, a recombinant soluble form of human CD39 based on the
structure of full-length CD39 was designed
(Figure 2
).16 CD39
contains 2 transmembrane regions near the amino and carboxyl termini,
respectively. These domains serve to anchor the native protein in the
cell membrane. Modeling studies, antibody epitope analyses, and
sequence homology have demonstrated that the portion of the molecule
between the transmembrane regions is external to the
cell.10 The extracellular
region contains the ACR characteristics of compounds in the apyrase
family, in concordance with the hypothesis that the external portion of
CD39 is critical for its ecto-ADPase activity. The ability of
CD39-expressing cells to metabolize extracellular
nucleotides supports the extracellular localization of the
enzymatic portion of the molecule. The fact that intracellular
nucleotide concentrations are maintained in the millimolar
range further suggests that the active site of CD39 is not exposed to
the cytoplasm.
For the generation of solCD39, the extracellular domain,
encoding 439 amino acids, was isolated with the use of
oligonucleotide cassettes and PCR, and was placed in a
mammalian expression
vector.16 Addition of the
interleukin-2 leader sequence ensured that the recombinant molecule was
secreted. After transfection with this solCD39-encoding plasmid, COS
cells generated levels of ATPase and ADPase activity in their
conditioned medium that linearly increased for a 5-day period. SolCD39
was isolated from conditioned medium derived from transiently
transfected COS cells via immunoaffinity chromatography
with the use of an anti-CD39 monoclonal antibody. This procedure
yielded a single
66-kDa protein with both ATPase and ADPase
activities, suggesting that the molecule had been properly glycosylated
in this cell system. Incubation of the purified protein with
N-glycanase to remove
N-linked
oligosaccharides yielded a band with the predicted molecular
weight of 52
kDa.16
Purified solCD39 blocks ADP-induced platelet aggregation
in vitro and also inhibits collagen-induced platelet
reactivity.16 Aggregation
induced by TRAP is also strongly blocked by CD39
(Figure 4
). This has led us to postulate that collagen and
TRAP depend more on released ADP for recruitment and aggregation than
was previously appreciated and underscores the possibilities of solCD39
as an antithrombotic agent.

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Figure 4. Inhibition and reversal of platelet aggregation. PRP from a donor who had ingested aspirin was stimulated with 5 µmol/L ADP, 2.5 µg/mL collagen (Chrono-Log), or TRAP as indicated. In vitro platelet responses to these agonists were strongly inhibited by abciximab and solCD39.
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 |
Site-Directed Mutagenesis Studies of Amino
Acids in the ACR Regions
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To identify amino acids critically important for the
enzymatic
and biological activity of CD39, we performed site-directed
mutagenesis
studies within the highly conserved ACR of solCD39.
Mutations
of Glu174 to Ala (E174A) and Ser218 to Ala (S218A) resulted
in
the loss of solCD39 enzymatic activity. Moreover, the Tyr127
to Ala
(Y127A) mutant lost 50% to 60% of ADPase and ATPase activity.
The
ADPase activity of wild-type solCD39 and of each mutant
was generally
greater with calcium than with magnesium, but
for ATPase activity, no
such preference was observed. Y127A
demonstrated the highest
calcium/magnesium ADPase activity ratio,
2.8-fold higher than that of
the wild type, although its enzyme
activity was greatly reduced.
Enzymatic activity always correlated
with biological activity in our
platelet aggregation system.
Thus, E174A, completely devoid of
enzymatic activity, failed
to inhibit platelet responsiveness.
S218A, with 91% loss of
ADPase activity, could still reverse
platelet aggregation, albeit
much less effectively than wild-type
solCD39. These data indicate
that Glu174 and Ser218 are essential for
both the enzymatic
and biological activity of solCD39, whereas Tyr127
appears to
play an important role as
well.
17
 |
Summary and Future Possibilities
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A CHO cellbased solCD39 expression system, capable of
growing
in a serum-free medium, has been developed to increase protein
production
and to facilitate protein purification. The
conditioned medium
from these CHO cells contains 20-fold more ATPase
and ADPase
activity than that obtained from COS
cells.
16 After the
administration
of this solCD39 preparation to mice, enzymatic activity
was
measurable for an extended period of time. The elimination phase
half-life
was

2 days.
16
Experimental results with a novel soluble form
of recombinant human
ecto-ADPase, solCD39, indicate a potential
for a new class of
antithrombotic agents acting by metabolism
of the ADP in an
activated platelet releasate. Thus, solCD39
blocks and
reverses platelet activation, preventing the recruitment
of
additional platelets into a growing thrombus. In this manner,
the
extent of occlusion as well as damage to the vascular wall
associated
with cardiac and cerebral vascular events, such as
stroke, myocardial
infarction, angioplasty, and stenting, can
largely be attenuated
(Figure 5

). Furthermore, because of its
independent mode of
action on the platelet releasate, solCD39
could be administered in
combination with currently used therapeutic
modalities, such as
heparin, aspirin, and glycoprotein IIb/IIIa
antagonists.

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Figure 5. Schematic depiction of thromboregulation by endothelial cell ecto-ADPase/CD39. Platelet activation on or proximal to a site of vascular injury induces release of ADP from platelet-dense granules (lower right inset). Released ADP activates and thereby recruits additional platelets, which have arrived in the local microenvironment of the evolving thrombus. Activation and recruitment of platelets in proximity to endothelial cells is inhibited by metabolism of released ADP to AMP by endothelial cell ecto-ADPase/CD39. CD39 does not act on the platelet per se but on the platelet releasate. These platelets then return to an unstimulated state, thereby limiting thrombus formation (upper left inset). Ecto-ADPase/CD39 has been identified and functionally characterized as a physiological constitutively expressed thromboregulator.
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Acknowledgments
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This study was supported in part by
Merit Review grants from
the Department of Veterans Affairs and by
National Institutes
of Health grants HL-47073, HL-46403, HL-59488,
NS-41462, and
NS-41460.
Received October 11, 2000;
accepted November 20, 2000.
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