A new role for the A2b adenosine receptor in regulating platelet function

ORIGINAL ARTICLE

A new role for the A2b adenosine receptor in regulating plateletfunction

D . Y A N G, * H . C H E N, *� M. KOUPE NOVA,* S . H . CARROLL ,* A . EL IADES ,* J . E . FREEDMAN,�P . TOSELL I* and K . RAV ID*�Departments of *Biochemistry and �Medicine, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA, USA

To cite this article: Yang D, Chen H, Koupenova M, Carroll SH, Eliades A, Freedman JE, Toselli P, Ravid K. A new role for the A2b adenosine

receptor in regulating platelet function. J Thromb Haemost 2010; 8: 817–27.

Summary. Background: Activation of platelets is a critical

component of atherothrombosis and plays a central role in the

progression of unstable cardiovascular syndromes. Adenosine,

acting through adenosine receptors, increases intracellular

cAMP levels and inhibits platelet aggregation. The A2a

adenosine receptor has already been recognized as a mediator

of adenosine-dependent effects onplatelet aggregation, andhere

we present a new role for the A2b adenosine receptor (A2bAR)

in this process. Methods and Results: As compared with

platelets from wild-type controls, platelets derived from

A2bARknockoutmice have significantly greaterADP receptor

activation-induced aggregation. Although mouse megakaryo-

cytes and platelets express low levels of the A2bAR transcript,

this gene is highly upregulated following injury and systemic

inflammation in vivo. Under these conditions, A2bAR-medi-

ated inhibition of platelet aggregation significantly increases.

Our studies also identify a novel mechanism by which the

A2bARcould regulate platelet aggregation; namely, ablationof

the A2bAR leads to upregulated expression of the P2Y1 ADP

receptor, whereas A2bAR-mediated or direct elevation of

cAMP has the opposite effect. Thus, the A2bAR regulates

platelet function beyond mediating the immediate effect of

adenosine on aggregation. Conclusions: Taken together, these

investigations show for the first time that the platelet A2bAR is

upregulated under stress in vivo, plays a significant role in

regulating ADP receptor expression, and inhibits agonist-

induced platelet aggregation.

Keywords: adenosine, platelets, receptors, stress, thrombosis.

Introduction

Atherothrombosis is a leading cause of mortality in the

Western world. Platelet accumulation at sites of vascular injury

is the primary event in arterial thrombosis, and the activation

of platelets is a critical component of atherothrombosis [1,2].

After the initial adhesion of platelets to extracellular matrix,

such as to collagen at sites of vascular injury, autocrine and

paracrine factors, including ADP, thrombin, epinephrine, and

thromboxane A2, amplify and sustain the initial platelet

response, and recruit circulating platelets to form a hemostatic

plug.Many agonists, such as ADP, thrombin, and epinephrine,

directly activate platelet Gi receptors and decrease the cAMP

level [3,4]. Other agonists, including collagen and thrombin,

also indirectly decrease the cAMP level by release of ADP [3]. It

is well established that cAMP is a critical cytosolic regulator of

platelet aggregation [3]. cAMP inhibits platelets by activating

protein kinase A (PKA), which phosphorylates several sub-

strates, such as inositol trisphosphate receptors, contributing to

inhibition of cytoskeletal reorganization, integrin activation,

and granule secretion [5].

Adenosine, an important platelet inhibitory mediator [6], is

also released from vascular wall cells and platelets into the

extracellular space as a breakdown product of ATP. Adeno-

sine, acting through adenosine receptors, stimulates G-protein-

coupled adenylyl cyclase in platelets and increases intracellular

levels of cAMP, which is a potent inhibitor of platelet

activation [6–8]. Platelets are rich in A2a adenosine receptors

(A2aARs) and, until recently, it has been speculated that the

A2aAR is the only adenosine receptor that significantly affects

platelet activation in response to adenosine [9]. A2aAR

knockout (KO) mice show higher platelet aggregation activity

in response to ADP [9]. It is well known that 5¢-N-ethyl-carboxamidoadenosine (NECA), an A2-type adenosine recep-

tor agonist, inhibits platelet activation [10,11]. However, the

A2aAR-selective agonist CGS 21680 [12] shows only 25–50%

of the effect of NECA, indicating another adenosine-mediated

effect, independent of A2aAR activation [8]. The A2b adeno-

sine receptor (A2bAR) is another Gs-coupled receptor [13]. A

recent study confirmed that A2bAR mRNA is expressed in

human platelets at similar levels to A2aARmRNA. This study

also employed pharmacologic ligands to detect an active

A2bAR on human platelets [14]. However, the importance of

the A2bAR has never been directly examined using a KO

approach, and has never been studied in vivo. Therefore, in the

Correspondence: Katya Ravid, CVI, W-601, Boston University School

of Medicine, Boston, MA 02118, USA.

Tel.: +1 617 638 5053; fax: +1 617 638 5339.

E-mail: [email protected]

Received 4 June 2009, accepted 11 January 2010

Journal of Thrombosis and Haemostasis, 8: 817–827 DOI: 10.1111/j.1538-7836.2010.03769.x

� 2010 International Society on Thrombosis and Haemostasis

present study, we combined genetic and pharmacologic

approaches to identify a new role for the A2bAR in platelet

function by using A2bAR KO/b-galactosidase (b-Gal) knock-

in mice generated in our previous study [15]. We also identified

a new mechanism by which this receptor affects platelet

aggregation. These investigations could lead to the develop-

ment of a new therapeutic approach to the control of

thrombosis by using specific A2bAR ligands.

Materials and methods

Animals

A2bAR KO/b-Gal knock-in mice were originally generated in

our laboratory [15]. In all experiments, C57BL/6J wild-type

(WT) (Jackson Laboratory, Bar Harbor, ME, USA) and KO

mice were matched for strain, sex, and age (10–12 weeks old).

All procedures were performed according to the Guidelines for

Care and Use of Laboratory Animals published by the NIH.

Throughout this study, all animals received humane care that

was in agreement with the guidelines of and approved by the

Institutional Animal Care and Use Committee of the Boston

University School of Medicine.

Platelet aggregation and shape studies

Aggregation was measured by light scattering in a platelet

aggregation profiler (Model PAP-4CD; BIO/DATA Corpora-

tion, Horsham, PA, USA), using platelet-rich plasma (PRP)

and washed mouse platelets, described in Doc. S1. Agonist-

induced changes in platelet shape were also followed by

electron microscopy, as detailed in Doc. S1.

Isolation and culture of mouse megakaryocytes (MKs)

Bone marrow cells were isolated and cultured as described

previously [16]. MKs were purified by the MACS magnetic

bead purification system (Miltenyi Biotech, BergischGladbach,

Germany), as described in [16] andDoc. S1. The purification of

MKs was confirmed by our recent study [17].

RNA isolation, cDNA synthesis and quantitative reverse-

transcription polymerase chain reaction (qRT-PCR)

RNA isolation and cDNA synthesis were performed as

described in Doc. S1. mRNA was quantified using TaqMan

Gene Expression Assays for P2Y1 receptor (P2Y1R) (assay

Mm00435471_m1; Applied Biosystems, Foster City, CA,

USA), with 18S rRNA as an endogenous control (Applied

Biosystems).

cAMP measurement in platelets and MKs

Washed platelets and MK-rich fractions were prepared as

described above. cAMP levels were measured by using a Direct

Cyclic AMP Enzyme Immunoassay Kit (Assay Designs, Inc.,

Ann Arbor, MI, USA), following the manufacturer�s instruc-tion. More details are given in Doc. S1.

Mouse femoral artery injury (FAI) model

WT and A2bAR KO male mice (10–12 weeks old) were

anesthetized and subjected to femoral artery wire injury, as

described in [18] and Doc. S1. At day 7 after injury, mouse

PRP was prepared and platelet aggregation was measured as

described above.

Lipopolysaccharide (LPS)-induced acute inflammation

WT or A2bAR KO mice were given a single intraperitoneal

injection of LPS (Sigma Aldrich, St Louis, MO, USA) at

5 lg g)1 body weight, or of saline in a total volume of up to

100 lL (control). Mice were euthanized 5 days post LPS or

saline administration, and blood was collected. PRP was

prepared as described above and used for platelet aggregation

studies.

b-Gal assay in MKs and platelets

MKs and platelets were prepared as described above. Cells

were fixed with 0.5% glutaraldehyde and stained with X-Gal,

as described in detail in [15] and Doc. S1. b-Gal expression in

platelets was analyzed at the ultrastructural level in the mouse

FAI model described above, by using a modification of a

previously published protocol [19].More details are provided in

Doc. S1.

Western blot analysis

Platelet protein isolation is described in Doc. S1. The mem-

branes were reacted with anti-vasodilator-stimulated phospho-

protein (VASP) (1 : 2000 dilution; Cell Signaling Technology

Inc., Danvers, MA, USA), which detects both the phosphor-

ylated and non-phosphorylated proteins, and reprobed with

anti-b-actin (1 : 10 000 dilution; Sigma-Aldrich) as loading

control.

Statistical analysis

Data are presented as average ± standard deviation. Two-

tailed Student�s t-test was used to compare two groups.

Differences were considered to be significant at P < 0.05.

Results

Higher aggregation activity of platelets derived from A2bAR

KO mice than of platelets with an active A2bAR

It has been speculated that the A2aAR is the only adenosine

receptor that significantly affects platelet activation in response

to adenosine [9], and in agreement with this, A2aAR KOmice

show higher platelet aggregation activity in response to ADP

818 D. Yang et al


[9]. Recently, an active A2bARwas detected in human platelets

[14]. First, we confirmed the presence of an active A2bAR in

mouse platelets, by measuring cAMP levels in WT mice.

NECA, which activates both the A2aAR and the A2bAR,

increased cAMP levels in platelets (Fig. 1A). The A2bAR-

selective antagonist MRS 1754 partly reversed the effect of

NECA (Fig. 1A), indicating that platelets express functional

A2bARs. cAMP levels measured after administration of both

NECA and MRS 1754 tended to be higher than in controls,

although the difference was not statistically significant. Because

of the relatively modest effect of NECA as compared with

controls, it is hard to detect the A2aAR-based activity left

following A2bAR antagonist treatment. The presence of an

active platelet A2aAR was confirmed by using an A2aAR-

selective agonist (Fig. 2C). This trend also existed in the platelet

precursors, the MKs, and, as anticipated, MRS 1754 did not

reverse the effect of NECA on cAMP in A2bAR KO cells

(Fig. S1). Finally, platelets derived from A2bAR KO mice

displayed reduced intracellular levels of cAMP, as well as of the

PKA-activated VASP [20] (Fig. 1B).

As cAMP is a critical cytosolic regulator of platelet

aggregation [3], and there are reduced levels of cAMP in

A2bARKO platelets, we determined the role of the A2bAR in

platelet aggregation by performing measurements under ago-

nist stimulation in WT and A2bAR KO cells. ADP (5 lM)-induced maximal platelet aggregation was 46% ± 8.7% in

WT mice vs. 60% ± 9.2% in A2bAR KO mice. Collagen

(10 lg mL)1)-induced maximal platelet aggregation was

57% ± 9.5% in WT mice vs. 74.8% ± 8% in A2bAR KO

mice. A significant increase (30–40%) was noted in A2bAR

KO samples as compared with WT samples (Fig. 2A,B).

Adenosine deaminase (1 U mL)1) did not induce significant

changes in either WT or A2bAR KO samples (data not

shown). The A2aAR-specific agonist CGS 21680 (which

elevates cAMP levels by about 1.5-fold; data not shown)

similarly inhibited ADP-induced platelet aggregation in both

WT and A2bAR KO mice (P > 0.05). This suggests that the

difference between WT and A2bARKO platelets is not due to

a change in A2aAR function (Fig. 2C). Importantly, the

platelet count is similar in WT and A2bAR KO mice [15].

Induction of expression of the A2bAR in platelets under stress

and its effect on the platelet aggregation response to NECA or

adenosine

In earlier studies, we showed that the expression of the A2bAR

is induced in smooth muscle cells by stresses, such as FAI or

LPS stimulation [18,21]. In our A2bAR KO/b-Gal knock-in

mouse model, the level of b-Gal activity reflects the expression

of the endogenous A2bAR gene previously. In an FAI model

in A2bAR KO mice, as reported in [18], b-Gal-positive

platelets were noted (Figs 3A and S2), indicating A2bAR gene

expression. A2bAR mRNA expression was also induced by

in vivo administration of LPS (Fig. S3). To further investigate

the significance of upregulated A2bAR expression under these

stress conditions, we compared the effects of NECA on ADP-

induced platelet aggregation in cells derived from femoral

artery injured or LPS-injected control WT and A2bAR KO

mice (Fig. 3B). ADP-induced platelet aggregation was inhib-

ited by NECA more effectively in WT samples obtained after

artery injury or LPS injection than in platelets derived from

A2bAR KO mice subjected to similar stresses, in which

minimal changes were noted (Fig. 3B). A comparable result

was obtained when adenosine was used instead of NECA

(Fig. 3C).

B

A40

987

65432

WT A2bAR KO

VASP

WT KO

β-Actin10

P < 0.05 P < 0.05

P < 0.05

cAM

P le

vel i

n pl

atel

ets

Bas

al c

AM

P le

vel i

n pl

atel

ets

(pm

ol m

g–1

prot

ein)

(pm

ol m

g–1

prot

ein)

Con

trol

NE

CA

MR

S 1

754

+ N

EC

A

MR

S 1

754

35

30

25

20

15

10

5

0

Fig. 1. A2b adenosine receptor (A2bAR) activation in mouse platelets.

(A) cAMP was measured in washed platelets derived from wild-type (WT)

mice as described inMaterials and methods after treatment with vehicle or

the A2-type adenosine receptor agonist 5¢-N-ethyl-carboxamidoadenosine

(NECA) (10 lM) for 10 min in the presence or absence of the A2bAR-

selective antagonist MRS 1754 (10 lM), as indicated. To prevent cAMP

degradation, the phosphodiesterase inhibitor paraverine hydrochloride

(200 microM) was added before NECA treatment. Data are presented as

average ± standard deviation (SD) (n = 4).P < 0.05 is considered to be

statistically significant. (B) cAMP and vasodilator-stimulated phospho-

protein (VASP) measurements in platelets derived from WT or A2bAR

knockout (KO) mice at baseline. Washed platelets were prepared as de-

scribed in Materials and methods. cAMP was measured in both WT and

A2bAR KO samples, following the manufactory instructions. Data are

presented as average ± SD (n = 5). P < 0.05 is considered to be sta-

tistically significant. Platelets from WT and A2bAR KO mice were also

subjected to western blot analysis (right side of the panel), using anti-

VASP and anti-b-actin as loading control. Protein kinase A phosphory-

lates VASP at serine 157, and as a result VASP shifts from 46 to 50 kDa in

sodium dodecylsulfate polyacrylamide gel electrophoresis [20]. Shown is a

representative experiment out of three.

A2b adenosine receptor and platelet activation 819


The P2Y1R is upregulated in A2bAR KO MKs and platelets

Platelets originate from polyploid MKs and thrombopoietin

(TPO) is the major mitogenic and differentiation regulator

of MK development and polyploidy. Considering the

inducible nature of the A2bAR gene, including by mitogens

[22], we first sought to determine its potential upregulation

by TPO. We found that this induction also applied to MKs.

There was a low level of A2bAR mRNA in primary mouse

MKs, which was increased in vitro and in vivo by TPO

treatment (Fig. 4A). This gene upregulation was further

demonstrated by staining for b-Gal in A2bAR KO MKs

(Figs 4B and S4). MK A2bARs are functional, as already

demonstrated in Fig. S1. The induction of A2bAR by TPO

had no effect on platelet counts (Fig. S5) or MK ploidy

(data not shown).

We previously showed that TPO upregulates the expression

of the ADP receptor P2Y1R in MKs [23], which is reminiscent

A

B

C

90

Min Min

Collagen

Ligh

t tra

nsm

issi

on (

%)

Ligh

t tra

nsm

issi

on (

%)

A2bAR KO

A2bAR KO

80

60

40

20

0

80

60

40

20

0ADP

WT

WT

P < 0.01

P > 0.05

P < 0.05

80

70

60

50

50

40

30

20

10

0

40

30

Pla

tele

t agg

rega

tion

(%)

CG

S 2

1680

inhi

bitio

n of

AD

P-

indu

ced

plat

elet

agg

rega

tion

(%)

WT A2bAR KO

WT A2bAR KO

ADPCollagen

20

10

0

Fig. 2. Platelet aggregation in wild-type (WT) and A2b adenosine receptor (A2bAR) knockout (KO) mice. Platelet-rich plasma was prepared, and

aggregation was measured as described in Materials and methods. (A) ADP (5 lM) and collagen (10 lg mL)1) induced platelet aggregation in WT and

A2bAR KO samples. Percentage aggregation is reflected by percentage increase in light transmission (wild type is set as 100%). A representative

aggregation profile is shown, for both the WT group and the A2bARKO group. (B) Data are presented as average ± standard deviation (SD) (n = 6 in

the ADP group, and n = 5 in the collagen group; P < 0.05 is considered to be statistically significant). There was a slight, but not significant, difference

betweenWT andKO platelets when the ADP concentration used inducedmild and high aggregation (at 1 or 10 lMADP). (C) Effect of the A2a adenosine

receptor agonist CGS 21680 (10 lM) on ADP-induced platelet aggregation (%) in WT and A2bAR KO samples. Data are presented as average ± SD

(n = 3). CGS 21680 was preincubated with platelets for 10 min, and this was followed by ADP (5 lM) treatment. The difference is not significant.

820 D. Yang et al


of its effect onA2bAR expression. Furthermore, using targeted

expression of the P2Y1R inMKs of transgenic mice, we found

that approximately two-fold upregulation of P2Y1R mRNA

level in platelets leads to increased ADP-induced platelet

aggregation [24], similar to the phenotype in A2bAR KO

platelets. Therefore, we considered a possible inverse correla-

tion between expression of the A2bAR and that of the P2Y1R

as an additional mechanism to explain the difference in platelet

aggregation response between WT and A2bAR KO mice.

Interestingly, it was found that P2Y1R expression was

A

B

C

60

lumen

WT A2bAR KO

lumen

1 µm

P < 0.05

P < 0.05

P < 0.05

50

40

30

20

45

35

25

15

50

40

30

20

10

10

NE

CA

inhi

bitio

n of

AD

P-in

duce

d

Ade

nosi

ne in

hibi

tion

of A

DP

-in

duce

d pl

atel

et a

ggre

gatio

n (%

)pl

atel

et a

ggre

gatio

n (%

)

Control

Control

FAI LPS

LPS

WT

A2bAR KO

WTA2bAR KO

0

Fig. 3. Effect of A2b adenosine receptor (A2bAR) activation on platelet aggregation. (A) Electron microscopic examination of platelets at 1 day

after femoral artery injury (FAI), a time point at which platelet adhesion is known to be displayed in this injury model [40]. In order to determine

b-galactosidase (b-Gal) expression in platelets of the A2bAR knockout (KO) mice (in which the deleted gene was replaced by the b-Gal gene), we used

electron microscopic examination. An arrow indicates platelets. The arrow head indicates A2bAR promoter-driven b-Gal expression (black signal)

in platelets. Original magnification: · 6400. As controls, injured wild-type (WT) mice were subjected to similar staining procedures, and show no

expression. Non-injured KO platelets show faint A2bAR expression (not shown). A larger picture is shown in Fig. S2. (B) Platelet-rich plasma (PRP) was

prepared from both WT and A2bAR KO mice subjected to non-FAI/LPS injection, FAI for 1 week and lipopolysaccharide (LPS) injection [5 lg g)1

intraperitoneal injection (i.p.)] for 5 days. ADP-induced platelet aggregation was measured as in Fig. 1 in the absence or presence of 5¢-N-ethyl-carboxamidoadenosine (NECA). NECA (10 lM)was preincubated with PRP for 2 min before addition of ADP (5 lM). The inhibition byNECAof ADP-

induced platelet aggregation was compared between groups (n = 4 in the FAI group, and n = 3 in the LPS injection group; P < 0.05 is considered to be

statistically significant). The absolute value is shown in Fig. S5. (C) The inhibition by adenosine of ADP-induced platelet aggregation in WT and A2bAR

KO mice. PRP was prepared from both WT and A2bAR KOmice subjected to saline and LPS injection (5 lg g)1 i.p.) for 5 days. ADP-induced platelet

aggregation wasmeasured as in Fig. 1 in the absence or presence of adenosine. Adenosine (10 lM)was preincubated with PRP for 2 min before addition of

ADP (5 lM). The inhibition by adenosine of ADP-induced platelet aggregation was compared between groups (n = 3; P < 0.05 is considered to be

statistically significant).



upregulated approximately two-fold in both A2bAR KO

platelets and MKs, as compared with WT cells (Fig. 5A). In

accordance with an upregulated P2Y1R in KO cells, the

frequency of platelets with ADP-induced shape changes was

greater in the KO samples than in the WT samples, as

demonstrated by electron microscopy (Fig. 5B,C). Taken

together, our findings suggest that intracellular changes in

cAMP levels in A2bAR KO cells, as well as consequent

changes in the expression of the P2Y1R, contribute to the

platelet aggregation phenotype in these mice.

B

A

4.5

3.54

32.5

21.5

10.5

A2b

AR

mR

NA

exp

ress

ion

in M

Ks

(fol

d)

A2b

AR

mR

NA

exp

ress

ion

in M

Ks

(fol

d)

0

4.5

3.5

MKs

P < 0.05

TPO-treated MKs

TPO injectionControl

4

32.5

21.5

10.5

0

β-Gal DAPI

WT

A2bAR KO

Fig. 4. Expression of the A2b adenosine receptor (A2bAR) gene in megakaryocytes (MKs) and platelets at baseline and under mitogenic stimulation.

(A) The upregulation of A2bAR mRNA expression by thrombopoietin (TPO) in vitro and in vivo. A2bAR mRNA expression was measured by

quantitative reverse-transcription polymerase chain reaction. In vitro study (upper panel): mRNA was prepared from freshly isolated MKs or MKs

cultured for 3 days , derived fromwild-type (WT) mice as in [16], purified with a CD41-based column as described inMaterials and methods. In vivo study

(lower panel): after treatment with TPO or phosphate-buffered saline (0.05 lg g)1, tail vein injection) for 4 days, the MKs were isolated and purified as

above. Data were normalized to 18S mRNA expression. (B) b-Galactosidase (b-Gal) staining of MKs derived from WT or A2bAR knockout (KO)

mice and viewed at · 600magnification with an Olympus IX70microscope combined with a Hamamatsu charge-coupled device camera (C4742-95). MKs

were isolated and cultured as in [16], and then subjected to b-Gal staining (seeMaterials andmethods). Blue staining inA2bARKOMKs (here displayed as

a darken shade) indicates the expression of b-Gal gene driven by the A2bAR gene promoter (left panel). 4¢,6-diamidino-2-phenylindole (DAPI) was used as

a nuclear counterstain (right panel). Bar: 5 lm. The lower-magnification picture is shown in Fig. S4.

822 D. Yang et al


B

C

A 3

2.5P < 0.05

P < 0.05

2

1.5

1

0.5

Platelets MKs

WT

A2bAR KO

WT

Control

ADP

A2bAR KO

WT+ ADP

A2bAR KO+ ADP

0

1 µm 1 µm

1 µm1 µm

Bas

al P

2Y1R

mR

NA

leve

l (fo

ld)

Fig. 5. Enhancement of megakaryocyte (MK) and platelet P2Y1 receptor (P2Y1R) expression and ADP-induced changes in platelets derived from

A2b adenosine receptor (A2bAR) knockout (KO) mice. (A) Quantitative reverse-transcription polymerase chain reaction was performed on RNA

extracted from washed platelets and CD41 column-purified MKs isolated from fresh bone marrow cells, as described in Materials and methods. P2Y1R

mRNA expression was measured and normalized to 18S mRNA expression. Data are presented as average ± standard deviation (SD) (n = 9 in the

platelet group, and n = 4 in the MK group). P < 0.05 is considered to be statistically significant. (B) ADP-induced platelet shape change in wild-type

(WT) and A2bAR KO mice. Transmission electronmicrographs of mouse platelets treated without ADP (resting platelets) or with ADP (activated

platelets) for 2 min and then fixed with glutaraldehyde are shown. Note the longitudinally sectioned and transversely sectioned platelets shown under

resting conditions [upper panels in (B)], depicted as round or elongated shapes, respectively, depending on the plane of sectioning. Arrows point to platelets

with a shape change following ADP stimulation. (C) There is about a two-fold increase in the frequency of cells with ADP-induced shape changes

(including platelet pseudopods) in the A2bARKO samples, as compared with ADP-activated WT platelets. Platelets with shape changes are indicated by

arrowheads. In agreement with this, there are fewer dense bodies (electron density depicted as dark intracellular dots) in the A2bAR KO ADP-activated

platelets than in the activated WT platelets. (C) is representative of eight different sections derived from each sample, with two mice analyzed per group.



cAMP downregulates P2Y1R mRNA expression in MKs

Next, we sought to explore the mechanism by which the

A2bAR might affect P2Y1R expression. As cAMP is a prime

mediator of A2bAR effects, we investigated whether A2bAR-

independent changes in cAMP level can also affect P2Y1R

mRNA expression. The results showed that both forskolin, a

direct activator of adenylyl cyclase, and 3-isobutyl-1-methyl-

xanthine (IBMX), a phosphodiesterase inhibitor, significantly

downregulated P2Y1R mRNA expression (Fig. 6). Further-

more, A2aAR activation by its selective ligand CGS 21680

downregulated P2Y1R mRNA expression (Fig. 6). This effect

was less pronounced than that observed with forskolin and

IBMX, and so was the effect on cAMP level (Fig. S1B).

These novel data demonstrate for the first time that cAMP

downregulates P2Y1R expression inMKs, and suggest a novel

role for A2-type adenosine receptors in the control of ADP

receptor expression, as further illustrated in Fig. 7.

Discussion

Platelet aggregation plays an important role in thrombus

formation, which is closely related to thrombotic disorders,

including atherosclerosis and arteriosclerosis, acute myocar-

dial infarction, angina, transient ischemic attacks and strokes,

and peripheral vascular diseases [3]. It is well known that

adenosine is an effective platelet aggregation inhibitor, and

until recently the A2aAR was believed to be the only

functional adenosine receptor mediating this effect [10,11,25].

In this study, we confirmed, for the first time, low-level

expression and activity of mouse platelet A2bAR that is

upregulated by mitogenic factors, including TPO, and by

vascular injury or inflammatory stimulation. We found that

platelets devoid of the A2bAR display a higher aggregation

response, which correlates with a reduction in the basal level

of cAMP in circulating platelets. Moreover, we report the

novel observation that activation of the A2bAR controls the

expression of ADP receptors and of ADP-induced platelet

aggregation.

The roles of adenosine as an inhibitor of platelet activation

and a stimulator of platelet cAMP levels are well established

[26]. However, the role of adenosine analogs as antithrombotic

agents is limited, mainly by their broad spectrum of pharma-

cologic effects. Knowledge of the subtypes of adenosine

receptors in platelets would be very useful in the design of

selective compounds for potential application in antithrom-

botic therapy. Coopper et al. [8] and others [10] have previously

shown that the A2aAR is involved in NECA-elicited increases

in cAMP levels in platelets, which play a role in platelet

activation. By using microarray analysis and real-time PCR,

Amisten et al. [14] recently detected a functional A2bAR in

human platelets. In our study, we used genetic and pharma-

cologic approaches, and further demonstrated the expression

and functional activity of the A2bAR in mouse MKs and

platelets. Coexpression of the A2aAR and the A2bAR in the

same cell has also been noted in other cell types, such as

vascular smooth muscle cells and macrophages [15,27,28]. It is

of note that aortic expression of the A2bAR is upregulated in

A2aAR null mice [29], whereas the level of the A2aAR appears

to be unchanged in A2bAR KO mice [15].

Platelet aggregation is a crucial biological system, and more

than one receptor subtype could represent a �second line of

defense�, as defined in [14]. As compared with the A2aAR, the

A2bAR is a low-affinity receptor [13], even though it exerts

the same intracellular second messenger cAMP effect. On the

one hand, expression of the A2bAR is inducible, and this

receptor could be important as an additional system in

situations of high adenosine levels, such as ischemia, or when

the receptor is transcriptionally induced, such as in cases of

injury or inflammation [18,21]. Hypoxia-inducible factor-1

and B-myb were shown to contribute to the upregulation of

the A2bAR under stress [22,30]. Both NADPH oxidase

(NOX)4 and tumor necrosis factor-a (TNF-a) are known to

be upregulated by inflammation and mitogenic stimuli [21].

As our earlier studies that showed an upregulating effect of

TNF-a and NOX4 on A2bAR expression [21], and consid-

ering our recent report on the expression of NOX in MKs

[17], we envision that stress-induced augmentation of NOX

and TNF-a induce A2bAR expression in MKs as well.

Future studies will examine these potential signaling media-

tors in this lineage. Consistent with our previous study and

investigations with other cell types and tissues [18,30–33], we

1.4

1.2

P < 0.05

P < 0.05

P < 0.05

P < 0.05

P < 0.05

Con

trol

For

skol

in

IBM

X

CG

S 2

1680

NE

CA

MR

S 1

754

+ N

EC

A

MR

S 1

754

1

0.8

0.6

0.4

0.2

P2Y

1R m

RN

A le

vel i

n M

Ks

(fol

d)

0

Fig. 6. cAMP downregulates P2Y1 receptor (P2Y1R) mRNA expression

in megakaryocytes (MKs). Quantitative reverse-transcription polymerase

chain reaction analysis of P2Y1R mRNA expression was performed on

wild-type MKs cultured for 4 days and stimulated with forskolin (2 lM),3-isobutyl-1-methylxanthine (IBMX) (10 lM), CGS 21680 (10 lM) or5¢-N-ethyl-carboxamidoadenosine (NECA) (10 lM) for 24 h. MKs were

pretreated with MRS 1754 (10 lM) for 10 min before NECA adminis-

tration. Data are presented as average ± standard deviation (n = 3 in

IBMXgroup, and n = 6 in all other groups).P < 0.05 is considered to be

statistically significant.

824 D. Yang et al


report here the inducibility of the A2bAR in the MK–platelet

system, raising the possibility that the platelet A2bAR could

become a significant subtype receptor during injury or

inflammation.

ADP is not only passively released from damaged erythro-

cytes and endothelial cells, but is also actively secreted from

platelet dense granules as the secondary activator after initial

aggregation induced by collagen, thrombin, or ADP itself

(reviewed in [34,35]). Two G-protein-coupled receptors medi-

ate platelet responses to ADP: the P2Y1R, which is coupled

with Gq and activates phospholipase C [36], and the P2Y12

receptor (P2Y12R), which is coupled with Gi and suppresses

cAMP formation [37]. Both pharmacologic studies and

investigations in P2Y1R KO mice showed the P2Y1R to be

necessary for the initiation of platelet aggregation in response

to ADP and to be essential in thrombotic states [36,38,39]. We

determined whether A2bAR ablation or activation is associ-

ated with changes in ADP receptor expression. Intriguingly,

the baseline level of P2Y1R mRNA expression is higher in

A2bAR KO MKs and platelets than in WT controls. This

could contribute to a different platelet aggregation response to

ADP. Indeed, in an earlier study, we showed that a mild

elevation in P2Y1R mRNA in MKs and platelets in vivo leads

to increased platelet aggregation [24]. We further confirmed

that elevated cAMP downregulates P2Y1R expression. It is,

then, conceivable that the lifelong lower basal cAMP level in

A2bAR KO platelets could contribute to greater basal

expression of ADP receptors, which would induce higher

aggregation activity. Future studies could focus on the

transcriptional or other regulation of P2Y1R by cAMP

modulation.

This study is novel in that it points to the induction of the

platelet A2bAR under stress, and to its role in inhibiting

platelet aggregation, associated with changes in cAMP level

and a newly identified regulation of the ADP receptor by

A2bAR-mediated changes in cAMP level (Fig. 7).

Injuryinflammation

(+)

(+)

(–)

(–)

(+)

(+)

A2bARA2bAR–/–

P2Y1R

Platelet

PlateletAdenylate cyclase

Platelet aggregation

cAMP

Gs

Fig. 7. Schematic presentation of the regulatory mechanism identified in this study. Stress-induced upregulation of the A2b adenosine receptor (A2bAR)

in the platelet lineage is associated with the ability of this receptor to significantly inhibit ADP-induced platelet aggregation; A2bAR activation can also

downregulate the level of the ADP receptor, the P2Y1 receptor (P2Y1R). (+) denotes upregulation and ()) denotes downregulation.



Addendum

The authors had full access to and take full responsibility for

the integrity of the data. All authors have read and agree to the

manuscript as written.

Acknowledgements

We thank B.M. Schreiber for insights on the vascular injury

model, L. Mycoff for assistance with the application of the

injury model and L. Beaulieu for initial assistance with

setting-up the platelet aggregation studies. This work was

supported by National Institutes of Health (NIH) Public

Health Services Grant HL13262. K. Ravid is an Established

Investigator with the American Heart Association. H. Chen is

supported by the National Institutes of Health Cardiovascular

Training Grant T32HL07224. M. Koupenova is supported by

the National Institutes of Health Cardiovascular Training

Grant HL007969.

Disclosure of Conflict of Interests

The authors state that they have no conflict of interest.

Supporting Information

Additional Supporting Informationmay be found in the online

version of this article:

Doc. S1. Methods.

Fig. S1. cAMP level in megakaryocytes (MKs) derived from

wild-type (WT) and A2bAR knockout (KO) mice.

Fig. S2. A higher magnification of the image in Fig. 3A,

depicting an electronmicroscopic examination of an artery and

platelets following femoral artery injury in A2bAR knockout

(KO) mice.

Fig. S3. Expression of the A2bAR gene in megakaryocytes

(MKs) at baseline and during inflammation.

Fig. S4. b-Galactosidase staining of cultured megakaryocytes

(MKs) derived from A2bAR knockout (KO) mice.

Fig. S5. Platelet counts before and after thrombopoietin (TPO)

injection in wild-type (WT) and A2bAR knockout (KO) mice.

Please note: Wiley-Blackwell are not responsible for the

content or functionality of any supporting materials supplied

by the authors. Any queries (other than missing material)

should be directed to the corresponding author for the article.

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A new role for the A2b adenosine receptor in regulating platelet function