From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
Purine Nucleotides Induce Regulated Secretion of von Willebrand Factor:
Involvement of Cytosolic Ca 21 and Cyclic Adenosine Monophosphate–Dependent
Signaling in Endothelial Exocytosis
By Ulrich M. Vischer and Claes B. Wollheim
von Willebrand factor (vWF) is stored and released from
endothelial secretory granules called Weibel-Palade (WP)
bodies. Acute release can be induced by thrombin, histamine, and other mediators of thrombosis or inflammation.
Their effect is thought to be mediated by an increase in
intracellular free calcium ([Ca21]i). Purine nucleotides such as
adenosine triphosphate (ATP) and adenosine diphosphate
(ADP) are released from platelet dense granules and from
ischemic tissues and are important regulators of platelet
function and vascular tone. In the present study, we investigated whether they could also induce exocytosis from
cultured endothelial cells. ATP (1 to 100 mmol/L) induced a
dose-related increase in vWF release, with a 2.3-fold maximal increase after 30 minutes. Similar responses were observed with ADP. ATP induced calcium mobilization from
intracellular stores, an effect mimicked by 2-methylthio-ATP,
a selective agonist for P2y receptors. However, 2-methylthioATP–induced vWF release was only 43% of the ATP response.
ATP-induced vWF release was also associated with a twofold
increase in cellular cyclic adenosine monophosphate (cAMP)
content, and was potentiated by 3-isobutyl-1-methylxanthine ([IBMX] added to increase cAMP levels by blocking
cellular phosphodiesterases) and 8-bromo-cAMP and inhibited by more than 50% by Rp-8-CPT-cAMPS, a competitive
protein kinase A inhibitor. Adenosine but not 2-methylthioATP mimicked the ATP-induced increase in cAMP. ATPinduced vWF release was partly inhibited by adenosine
deaminase, which degrades adenosine generated from ATP
in the incubation medium. Adenosine (1 to 100 mmol/L)
failed to induce vWF release, but potentiated the secretory
response to 2-methylthio-ATP and thrombin without modifying the calcium response to these agents. Our results suggest that ATP/ADP can induce vWF release from endothelial
cells via dual activation of P2y and adenosine A2 receptors.
ATP/ADP-induced exocytosis could be involved in the regulation of thrombus formation and ischemia-reperfusion injuries. Further, we provide evidence that a receptor-mediated
increase in cellular cAMP can potentiate the secretory response to calcium-mobilizing agents.
r 1998 by The American Society of Hematology.
T
complement components C5a and C5b-9,10,11 leukotrienes,12
and superoxide anions.13 The principal recognized intracellular
signaling event involved in the response to these activators is a
rapid increase in cytosolic free calcium ([Ca21]i) and the
consequent activation of calmodulin-dependent processes.7,14 In
addition, we have recently observed that in vitro, epinephrine
can induce vWF release and potentiate the secretory response to
thrombin, a well-characterized calcium-mobilizing agonist. The
effect of epinephrine was associated with an increase in cyclic
adenosine 38,58 monophosphate (cAMP), providing evidence
that cAMP-dependent signaling can modulate exocytosis of WP
bodies.15
Extracellular purine nucleotides may play an important
physiologic role in the regulation of vascular tone and platelet
function.16 Extracellular adenosine triphosphate (ATP) and
adenosine diphosphate (ADP) are released at high concentrations from platelet dense granules.17 The main additional source
is ischemic tissue. Indeed, ATP is released from perfused
myocardium after only short periods of coronary occlusion.18
ATP or ADP can modulate vascular tone via regulation of the
production of prostacyclin and nitric oxide from endothelial or
smooth muscle cells.16 ADP is an important activator of platelet
aggregation.16 Its key role in hemostasis is suggested by the
bleeding tendency observed in patients with several forms of
storage pool disorders, associated with impaired ADP release
from platelet dense granules.19 In endothelial cells, ATP or ADP
binds to specific membrane receptors, which induces an increase in [Ca21]i. Several receptor subtypes have been described and are classified according to the relative potency of
ATP-related agonists.16 The P2y subtype, activated by 2methylthio-ATP . ATP 5 ADP . uridine triphosphate, seems
to predominate in human endothelial cells, although expression
of additional subtypes has not been excluded.20 Both in vivo and
in vitro, ATP is rapidly converted to ADP, AMP, and adenosine
through sequential reactions catalyzed by ecto-ATPases and 58
HE VASCULAR endothelium plays a central role in blood
coagulation and the inflammatory response. Upon activation, endothelial cells acquire a procoagulant state and also
become adhesive for circulating leukocytes, which allows their
migration to the extravascular space. One important activation
mechanism is agonist-induced exocytosis.1 By this process,
preformed secretory granules fuse with the plasma membrane.
These granules, called Weibel-Palade bodies (WP bodies), store
and release von Willebrand factor (vWF), an adhesive glycoprotein involved in primary hemostasis.1,2 Indeed, vWF is essential
for platelet adhesion to the vascular subendothelium, at least
under conditions of high shear stress.3 Exocytosis of WP bodies
also induces P-selectin translocation to the cell membrane. This
granule membrane protein is an adhesion molecule that mediates rolling and subsequent extravasation of circulating leukocytes.4 Expression of P-selectin at the cell surface is thought to
play a major role in the inflammatory response4 and in
neutrophil-mediated damage in ischemia-reperfusion injuries.5,6
In cultured endothelial cells, WP bodies can release their
contents after stimulation with thrombin,7 histamine,8 fibrin,9
From the Division de Biochimie Clinique, Department of Medicine,
Centre Médical Universitaire, Geneva, Switzerland.
Submitted January 16, 1997; accepted August 12, 1997.
Supported by Grants No. 32-41941.94 (U.M.V.) and 32-32376.91
(C.B.W.) and a SCORE subsidy (U.M.V.) from the Swiss National
Science Foundation.
Address reprint requests to Ulrich M. Vischer, MD, Division de
Biochimie Clinique, CMU, 1 rue Michel Servet, 1211 Geneva 4,
Switzerland.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1998 by The American Society of Hematology.
0006-4971/98/9101-0009$3.00/0
118
Blood, Vol 91, No 1 (January 1), 1998: pp 118-127
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
ATP/ADP-INDUCED vWF SECRETION
nucleotidase.21 The biologic functions of ATP can therefore be
modulated by the actions of its metabolites, particularly adenosine. Adenosine A2 receptors are expressed in endothelial and
other vascular cells, and mediate the pleiotropic biologic
functions of adenosine via activation of cAMP-dependent
signaling pathways. They are involved in the regulation of
vascular tone, endothelial permeability, and neutrophil activation.22
Since ATP is a calcium-mobilizing agent, via activation of
P2y receptors, it could be predicted to induce exocytosis from
endothelial cells. However, a previous report found that ATP is
only a weak stimulator of vWF release from cultured human
umbilical vein endothelial cells (HUVECs), in apparent contradiction to the central role of calcium in the regulation of
endothelial exocytosis.23 In the present study, we investigated
the role of purine nucleotides in exocytosis assessed by acute
vWF release and characterized the associated intracellular
signaling events.
MATERIALS AND METHODS
Materials. RPMI 1640 medium was obtained from GIBCO-BRL
(Gaithersburg, MD), and fetal calf serum (FCS) and collagenase were
from Seromed (Berlin, Germany). Endothelial Cell Growth Supplement
(ECGS) was from Upstate Biotechnology Inc (Lake Placid, NY).
Anti-vWF antibodies were from Dako (Glostrup, Denmark). ATP,
adenosine, ADP-bS, and adenosine deaminase (ADA) were from Fluka
(Buchs, Switzerland). 2-Methylthio-ATP and 2-chloro-ATP were from
RBI (Natick, MA). UTP, human thrombin, 3-isobutyl-1-methylxanthine
(IBMX), and forskolin were from Sigma (St Louis, MO). Rp-8-CPTcAMPS was from Biolog (Bremen, Germany). Fura-2 acetoxymethylester (fura 2-AM) was from Teflabs (Austin, TX), and ionomycin was
from Calbiochem (La Jolla, CA).
Cell culture. Primary cultures of endothelial cells (HUVECs) were
obtained from individual human umbilical veins by collagenase digestion as described previously.13 They were grown in RPMI 1640 medium
supplemented with 10% FCS, 90 µg/mL heparin, and 15 µg/mL ECGS.
Cells were used during passages 1 to 3. The tissue culture dishes,
24-well plates (Costar, Cambridge, MA), and glass coverslips were
coated with 0.1% gelatin.
Secretion studies. Confluent monolayers of HUVECs grown in
24-well dishes were washed three times and preincubated in 1 mL
Krebs-Ringer bicarbonate buffer (120 mmol/L NaCl, 4.75 mmol/L KCl,
1.2 mmol/L KH2PO4, 0.6 mmol/L MgSO4, 1.2 mmol/L CaCl2, 25
mmol/L NaHCO3, and 25 mmol/L HEPES, pH 7.4 [KRBH], supplemented with 0.1% BSA) for 10 minutes at 37°C. After a fourth wash,
cells were incubated in 0.3 to 0.5 mL KRBH with the different agents.
All pharmacologic agents were directly dissolved in incubation medium; only forskolin and IBMX were dissolved in dimethylsulfoxide
(DMSO). The final concentration of DMSO in the incubation medium
did not exceed 0.2%, a level that has no effect on vWF release.
To control for cell lysis, the activity of the cytosolic enzyme lactate
dehydrogenase was measured fluorometrically as previously described.13
vWF measurements. vWF levels were measured by enzyme-linked
immunosorbent assay (ELISA) as described previously.13 A standard
curve was constructed from serial dilutions of normal pooled plasma
assuming a plasma concentration of 10 µg/mL. Results are usually
expressed in nanograms per well per time unit. We observed considerable variation in the cellular vWF content and rate of secretion between
cell batches. When necessary, the results are therefore expressed in
119
relative values, ie, as a percentage of release from unstimulated control
cells from the same cell preparation. Unless indicated otherwise, results
are shown as the mean 6 SEM. Statistical analysis was performed using
the two-tailed, paired Student’s t-test.
vWF immunoprecipitation and sodium dodecyl sulfate–agarose gel
electrophoresis. To examine the multimeric composition of released
vWF, HUVECs were metabolically labeled with 50 µCi/mL [35S]cysteine and [35S]-methionine (PromixTM; Amersham, Little Chalfont,
UK) for 24 hours in methionine and cysteine–deficient RPMI 1640. The
cells were then stimulated with various agonists as already described.
The supernatants and labeling medium were supplemented with Tris (50
mmol/L, pH 8.0), EDTA (2 mmol/L), phenylmethylsulfonyl fluoride (1
mmol/L), iodoacetic acid (1 mmol/L), and N-methyl-maleimide (1
mmol/L). vWF was immunoprecipitated from the samples with an
anti-vWF antiserum preadsorbed to protein A–Sepharose, and was
resolved on a nonreducing 2% sodium dodecyl sulfate (SDS)-agarose
gel as previously described.24 The gel was processed using a phosphorImager (Molecular Dynamics, Sunnyvale, CA).
Measurement of [Ca21]i. HUVECs grown on glass coverslips were
loaded with 1 µmol/L fura 2-AM in culture medium (containing 10%
FCS) for 30 to 60 minutes at 37°C. The coverslips were rinsed,
incubated for 5 to 10 minutes in KRBH at room temperature, and
immersed in a glass cuvette containing 1.5 mL KRBH under constant
stirring. The cuvette was held at constant temperature (37°C). Fluorometric readings were performed with a Jasco (Hachiogi City, Japan)
CAF-110 fluorimeter. The fluorescent excitation was 340 and 380 nm,
and the emission wavelength was 490 nm. The [Ca21]i was calculated
according to the equation of Grynkiewicz et al25: [Ca21]i 5 KD 3 B 3
(R 2 Rmin)/(Rmax 2 R), where R is the ratio of fura-2 fluorescence at
340 nm excitation divided by the fluorescence at 380 nm excitation,
Rmax is the ratio when all fura-2 is bound to calcium, Rmin is the ratio
when all fura-2 is in the free form, B is the ratio of the fluorescence of
free fura-2 divided by the fluorescence of calcium-bound fura-2 with
380 nm excitation, and KD is the dissociation constant of calcium
binding to fura-2 (224 nmol/L). Rmax was measured at the end of the
recording after addition of ionomycin 10 µmol/L and CaCl2 to a final
concentration of 3.2 mmol/L. EGTA 8 mmol/L and Tris 40 mmol/L, pH
9.0, were then added for measurement of Rmin.
cAMP measurements. Confluent HUVECs grown in culture dishes
(diameter, 35 mm) were handled for secretion studies as already
described. At the end of the incubations, the cell monolayer was
extracted with 0.6 mL ice-cold ethanol (70% vol/vol). After separation
from proteins by centrifugation, the extract was dried in a Speedvac and
reconstituted in assay buffer. cAMP levels were measured by radioimmunoassay using a commercial kit (Amersham).
RESULTS
ATP and ADP induce vWF release via the regulated secretory
pathway. To investigate the effects of extracellular adenosine
nucleotides on exocytosis from endothelial cells, we incubated
confluent HUVECs with ATP, ADP, and thrombin for 30
minutes. vWF release was measured by ELISA in the supernatant (Fig 1). We observed a dose-related increase in vWF
release, with a 2.3-fold increase (from 1.9 1 0.3 to 4.3 1 0.7
ng/well/30 min, P , .02, n 5 8) with ATP and a 2.4-fold
increase (from 2.0 6 0.3 to 4.9 6 1.1 ng/well/30 min, P 5 .04,
n 5 5) with ADP at concentrations of 100 µmol/L (Fig 1A and
B). The response to ATP or ADP was smaller than the response
to thrombin, which induced a 4.6-fold increase (from 2.0 6 0.3
to 9.3 6 1.2 ng/well/30 min, P , .02, n 5 5) at a concentration
of 1 U/mL (Fig 1C). Incubation with IBMX, a phosphodiester-
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
120
VISCHER AND WOLLHEIM
Fig 1. Effect of ATP and ADP on vWF release. Confluent HUVECs grown in 24-well plates were incubated for 30 minutes at 37°C at the
indicated concentrations of ATP (A), ADP (B), and thrombin (C) in either the absence (N) or the presence (X) of IBMX (100 mmol/L). vWF release
was measured in the supernatant by ELISA. Results are the mean 6 SEM of 8 (A) and 5 (B and C) experiments. *P F .05 v unstimulated control
cells.
ase inhibitor added to prevent cAMP degradation, failed to
induce vWF release, as previously reported.15 However, in the
presence of IBMX, the response to ATP and ADP was much
stronger, with a 5.9- and 5.7-fold increase, respectively (P , .05),
and was detectable at nucleotide concentrations as low as 1
µmol/L. In contrast, IBMX had only a minor potentiating effect
on the response to thrombin. Thus, in the presence of IBMX, the
responses to ATP, ADP, and thrombin were similar (Fig 1A v C).
vWF released from WP bodies consists of high–molecular
weight multimers, whereas constitutively released vWF consists predominantly of dimers and small multimers.24 We
therefore determined the multimer pattern of vWF released in
response to ATP (Fig 2). Confluent HUVECs were incubated for
24 hours with [35S]-labeled methionine and cysteine. After
stimulation with ATP, ATP/IBMX, or thrombin for 30 minutes,
released vWF was immunoprecipitated from the supernatant and
resolved on a nonreducing SDS-agarose gel. vWF released from
cells stimulated with ATP (6 IBMX) consisted of high–
molecular weight forms in a pattern similar to the one observed
after stimulation with thrombin. In contrast, vWF from unstimulated cells consisted of a small amount of dimers. vWF
constitutively released over 24 hours into the culture medium
during the labeling period also consisted predominantly of
dimers and low–molecular weight multimers. These results
suggest that ATP induces vWF release from WP bodies, ie, via
regulated secretion. This conclusion was confirmed by immunofluorescence experiments. ATP stimulation caused a decrease in
the number of WP bodies and the appearance of extracellular
deposits of vWF associated with the subendothelium, typical of
regulated secretion.13,24 Again, the pattern was similar to that
observed after stimulation with thrombin (data not shown).
We next performed a time-course study of ATP-induced vWF
release (Fig 3). Release from cells stimulated with 100 µmol/L
ATP was unchanged as compared with control cells after 5
minutes. However, there was a 2.9-fold increase after 10
minutes (P , .02), which continued during the 60 minutes of
the study. Although the increase in vWF release was larger,
addition of IBMX had no significant effect on the lag time of the
ATP response. In contrast, the response to thrombin was much
faster, with a 3.9-fold increase over control cells already after 5
minutes (P , .05). IBMX had no significant effect on thrombin-
induced vWF release after 5 and 10 minutes, with a minor
potentiating effect becoming obvious only after 30 minutes.
Thus, ATP-induced vWF release (although delayed when
compared with thrombin) occurs in less than 30 minutes, again
suggesting release from preformed stores.
Fig 2. Multimer pattern of vWF released in response to ATP.
Confluent HUVECs were metabolically labeled with [35S]-cysteine and
[35S]-methionine for 24 hours. vWF was immunoprecipitated from the
labeling medium (medium) and from the supernatant of cells incubated for 30 minutes with ATP (100 mmol/L), ATP/IBMX (100 mmol/L),
thrombin (1 U/mL), or KRBH-BSA 0.1% alone (control). vWF multimers were resolved on a horizontal nonreducing 2% SDS-agarose gel.
Top arrowhead, the origin of the gel; bottom arrowhead, position of
vWF dimers. vWF released constitutively into the labeling medium
consists predominantly of dimers and small multimers, whereas vWF
released in response to ATP and thrombin consists of high–molecular
weight multimers.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
ATP/ADP-INDUCED vWF SECRETION
Fig 3. Time course of ATP-induced vWF release. Confluent HUVECs grown in 24-well plates were incubated for the indicated times
with 100 mmol/L ATP, ATP/IBMX (both 100 mmol/L, ATP/I), 1 U/mL
thrombin (Thr), or 1 U/mL thrombin and 100 mmol/L IBMX (Thr/I).
Control cells were incubated with KRBH-BSA 0.1% alone (Contr) or
supplemented with 100 mmol/L IBMX (I). Results are the mean 6 SEM
of 5 experiments; error bars were omitted for the sake of clarity. The
SEMs were less than 25% of the mean of all values.
Involvement of P2y receptor activation in vWF release. The
slower time course and the modulation by IBMX suggest that
ATP induces vWF release through signaling mechanisms at
least in part distinct from those elicited by thrombin. To
characterize the receptor subtype(s) involved, we studied vWF
121
release in response to receptor-specific pharmacologic agonists.
Defining the secretion produced by ATP (100 µmol/L for 30
minutes) as 100%, the response to UTP was negligible (17 6 6%,
NS, n 5 4), arguing against the involvement of a P2u receptor
in vWF release. The response to the P2y agonists 2-chloro-ATP,
MeS-ATP, and ADP-bS (all tested at 100 µmol/L) was 126% 6
16%, 43% 6 5% and 48% 6 9%, respectively (n $ 4, P , .05
v unstimulated controls for all three agonists). These results
suggest that P2y activation elicits vWF release. However, the
relative potency of the agonists, in particular the weaker
responses to 2-methylthio-ATP and ADP-bS, suggest that P2y
activation does not fully account for ATP-induced vWF release.
Implication of adenosine in the ATP response. Both in vivo
and in vitro, ATP is rapidly converted to AMP and adenosine
through sequential reactions catalyzed by ectonucleotidases.16,21 We therefore wondered whether the effect of ATP
could be mediated by adenosine (Fig 4A). Adenosine added
alone had no effect, but when added together with IBMX it
induced a 2.2-fold increase (P , .05) in vWF release. However,
2-methylthio-ATP and adenosine added together induced a
secretory response comparable to that of ATP. The synergistic
effect of adenosine and 2-methylthio-ATP was observed in both
the absence and presence of IBMX. This observation suggests
that the response to ATP may result from activation of both P2y
and adenosine receptors. This interpretation implies that adenosine is generated from ATP in the incubation buffer in concentrations sufficient to activate its receptor. We performed a doseresponse study of the effect of adenosine on 2-methylthio-ATP–
induced vWF release (Fig 4B). In the absence of IBMX, we
observed a maximal response to adenosine at 10 µmol/L, with a
significant effect already seen at 1 µmol/L. The dose-response
profile was similar in the presence of IBMX, except for a larger
potentiating effect at high adenosine concentrations (100 µmol/
L). Thus, adenosine produced by conversion of less than 10% of
Fig 4. Synergistic effect of adenosine and 2-methylthio-ATP on vWF release. (A) Confluent HUVECs were incubated for 30 minutes at 37°C in
the presence of ATP adenosine (Ado), and 2-methylthio-ATP (MeS) (all agonists tested at 100 mmol/L) in the presence or absence of 100 mmol/L
IBMX. Results are the mean 6 SEM of 5 experiments. Because of large variations in vWF release in this series, the results are shown as relative
values, with release at 30 minutes from unstimulated cells defined as 100%. The effects of MeS and Ado alone were small, but the response to the
combination of Mes 1 Ado was similar to or greater than the response to ATP. *P F .02 v control (C) cells; **P F .02 v control cells and cells
treated with Ado or MeS alone. (B) Adenosine-induced vWF release: dose-response in the presence of IBMX (100 mmol/L), MeS (100 mmol/L), and
IBMX 1 MeS. Each combination was added to confluent HUVECs for 30 minutes at 37°C. Results are the mean 6 SEM of 5 experiments. *P F .002
v control cells not stimulated with adenosine.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
122
VISCHER AND WOLLHEIM
the added ATP is sufficient to potentiate the secretory response
to activation of P2y receptors.
To confirm this contention, we added ADA to the cells to
degrade any adenosine produced during the incubation with
ATP (Fig 5). ADA (1 U/mL) inhibited the ATP response by
23% 6 3% and 52% 6 3% in the presence and absence of
IBMX, respectively (P , .001, n 5 8). Higher concentrations
of ADA failed to achieve greater inhibition. The secretory
response could be reconstituted by addition of 2-chloroadenosine, an adenosine analog resistant to ADA. These results
confirm that ATP-induced vWF release is mediated in part by
adenosine, most likely generated by partial degradation of the
added ATP. The inhibition by ADA and rescue by 2-chloroadenosine suggest that adenosine itself, rather than inosine or
other adenosine degradation products, is involved. Indeed,
when adenosine and 2-chloro-adenosine were tested in parallel,
together with IBMX and/or 2-methylthio-ATP, the effect of
2-chloro-adenosine was always equal to or stronger than the
effect of adenosine itself (not shown).
Involvement of cAMP in ATP-induced vWF release. Activation of P2y receptors by ATP or ADP is known to be associated
with an increase in [Ca21]i due to calcium mobilization from
intracellular stores.20 Numerous reports have convincingly
Fig 6. Effect of protein kinase A inhibition on ATP-induced vWf
release. Confluent HUVECs grown on 24-well plates were incubated
for 30 minutes in the presence or absence of Rp-8-CPT-cAMPS (Rp).
ATP and/or IBMX were then added, and the incubation continued for
30 additional minutes. Results are expressed as vWf released per well
during the last 30 minutes. Basal release was calculated as half the
vWF released during the 1-hour incubation (assuming continuous
constitutive release over 1 hour, see Fig 2). See text for statistical
analysis.
Fig 5. ATP-induced vWF release: Effect of adenosine removal by
ADA. Confluent HUVECs were incubated with ADA (1 U/mL) 6
2-chloro-adenosine (2Cl, 10 mmol/L) as indicated; 3 minutes later, 100
mmol/L ATP was added where indicated, and the incubation continued for 30 minutes at 37°C. The experiment was performed in the
absence or presence of 100 mmol/L IBMX. Data are the mean 6 SEM
of 5 to 8 experiments. *P F .05.
demonstrated the key role of cytosolic calcium in the regulation
of vWF release from WP bodies. However, the relative efficacy
of ATP and 2-methylthio-ATP, ADP-bS, and 2-chloro-ATP
indicates that the activation of P2y receptors is a poor stimulus
for vWF release and suggests the participation of additional
messenger systems. We tested the involvement of the cAMP
signaling pathway in ATP-induced vWF release using Rp-8-CPTcAMPS, a competitive inhibitor of protein kinase A (Fig 6). In
the presence of Rp-8-CPT-cAMPS (500 µmol/L), basal vWF
release was inhibited by 47% 6 11% (P , .05, n 5 4). Even
when the lower basal release was taken into account, ATPinduced vWF release was inhibited by 49% 6 7% and the
response to ATP/IBMX was inhibited by 71% 6 7% (P , .05,
n 5 4). vWF release in response to forskolin, used as a control
activator of the cAMP pathway, was reduced by greater than
50% (not shown). Lower concentrations of the inhibitor were
ineffective. The related inhibitors Rp-cAMPS and Rp-MBcAMPS had only weak inhibitory effects on the response to
either ATP or forskolin, in keeping with their lower lipophilicity.
To confirm the involvement of cAMP in vWF release, we
tested the effect of 8-bromo-cAMP, a cell-permeant cAMP
analog. Cultured HUVECS were incubated for 40 minutes with
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
ATP/ADP-INDUCED vWF SECRETION
123
8-bromo-cAMP (1 mmol/L), and additional agonists were
added after the first 20 minutes. In the presence of 8-bromocAMP (1 mmol/L) added alone, vWF release increased from
1.5 6 0.2 to 1.9 6 0.3 ng/well/20 min (nonsignificant, P 5 .14,
n 5 5). However, vWF release in response to ATP increased
from 4.4 6 0.8 to 10.0 6 2.4 ng/well/20 min (1308%), to
2-methylthio-ATP from 3.1 6 0.5 to 6.4 6 1.5 ng/well/20 min
(1344%), and to thrombin from 7.3 6 1.2 to 12.8 6 2.3
ng/well/20 min (1196%) (P , .01 for all three comparisons).
Thus, 8-bromo-cAMP can potentiate the secretory response to
several calcium-mobilizing agonists.
To better define the involvement of cAMP in ATP-induced
vWF release, in particular to identify the receptor involved, we
measured cellular cAMP content by radioimmunoassay (Table
1). ATP (100 µmol/L) added for 30 minutes caused a 2.0-fold
increase in cAMP content (from 1.10 6 0.19 to 2.23 6 0.55
pmol/plate, P , .05, n 5 5). Incubation with IBMX caused a
1.7-fold increase in cAMP content (P , .05, n 5 5), while ATP
added together with IBMX caused a 3.1-fold increase. This
observation confirms that the potentiation of IBMX on ATPinduced vWF release is due to its effect on cAMP levels. The
effect of ATP on cAMP content was mimicked by stimulation
with adenosine (P , .05) but not with 2-methylthio-ATP
(P 5 .19) (both agents tested at 100 µmol/L). Similar results
were observed in the presence of IBMX (P 5 .01 and .07,
respectively). These results indicate that the cAMP-mediated
potentiation of ATP-induced vWF release involves activation of
adenosine receptors. The effect of adenosine or ATP on cellular
cAMP content is equal to if not greater than that of epinephrine,
a known receptor-mediated activator of adenylate cyclase.
cAMP levels are minimally increased by 2-methylthio-ATP and
thrombin, and were only marginally increased in response to
thrombin. It is therefore highly unlikely that the effect of these
calcium-mobilizing agents directly implicates activation of a
calcium-sensitive adenylate cyclase.
Effect of ATP and adenosine on [Ca21]i. P2y receptors are
seven-transmembrane domain receptors that induce IP 3Table 1. Effect of ATP, Adenosine, and 2-Methylthio-ATP
on Cellular cAMP Content
cAMP Content (fmol/cm2)
Agonist
Control
ATP (100 µmol/L)
Adenosine (100 µmol/L)
2-Methylthio-ATP (100 µmol/L)
2-Methylthio-ATP (100 µmol/L 1 adenosine
(100 µmol/L)
Thrombin (1 U/mL)
Epinephrine (1 µmol/L)
Forskolin (10 µmol/L)
Without
IBMX
With IBMX
(100 µmol/L)
123 6 21
247 6 61*
221 6 28*
162 6 19
203 6 40
385 6 57*
378 6 54*
228 6 31
281 6 23*
175 6 26
—
—
440 6 59*
280 6 42*
348 6 37*
885 6 113*
HUVECs grown on culture dishes (35 mm) were handled in the same
way as in the secretion studies. Incubations were performed for 30
minutes at 37°C in the presence of agonists as indicated. cAMP was
extracted in 70% ethanol and measured by radioimmunoassay. Results are expressed as the mean 6 SEM of 4 individual experiments.
vWF release was always measured in the supernatant, with results
entirely consistent with the data in Figs 1 and 3.
*P , .05 versus corresponding control.
Fig 7. Effect of ATP and adenosine on [Ca21]i. HUVECs were grown
on gelatin-coated glass coverslips, loaded with fura-2, and immersed
in a quartz cuvette for radiometric fluorescent emission measurements. Each tracing shown is representative of at least 3 similar
experiments. (A) Stimulation with 100 mmol/L ATP was followed by
0.5 U/mL thrombin (Thr). The [Ca21]i response to thrombin looks
virtually identical when tested alone, and is not modified by prior
stimulation with ATP (not shown). (B and C) The response to
2-MeS-ATP was similar to the ATP response (B), and was not modified
by preincubation with IBMX and adenosine (Ado), both tested at 100
mmol/L (C).
mediated calcium mobilization from intracellular stores.16,20 To
confirm the involvement of P2y receptors in ATP-induced vWF
release, we performed measurements of [Ca21]i using the
fluorescent calcium-sensitive probe fura-2. HUVECs were
grown on glass coverslips, loaded with fura-2, immersed in a
cuvette, and stimulated with ATP, adenosine and MeS-ATP, and
thrombin (Fig 7). Thrombin induced a rapid but transient
increase in [Ca21]i (with a peak elevation from 79 6 20 to
399 6 43 nmol/L, n 5 3), followed by a second phase of more
stable elevation, with a mean value of 130 6 25 nmol/L after 3
minutes. ATP caused a similar but smaller initial elevation (from
58 6 16 to 279 6 34 nmol/L), but with a maximal amplitude
that was only 70% of that observed after thrombin. [Ca21]i
values returned rapidly to near baseline (79 6 20 nmol/L after 3
minutes; Fig 7A). IBMX failed to alter the [Ca21]i profile in
response to ATP (not shown). An almost identical profile was
observed in response to MeS-ATP (100 µmol/L), with a peak
elevation from 83 6 21 to 263 6 3 nmol/L (n 5 3; Fig 7B).
These results are compatible with P2y receptor–mediated
mobilization of calcium from an intracellular store, as reported
in detail previously.20 Adenosine had no effect on [Ca21]i even
in the presence of IBMX (Fig 7C). The [Ca21]i response to
2-methylthio-ATP was not modified by preincubation with
adenosine and IBMX as compared with 2-methylthio-ATP
alone (maximal elevation from 68 6 2 to 268 6 60 nmol/L,
n 5 3; Fig 7C). This last observation was confirmed with longer
recordings of up to 10 minutes (not shown). These results
strongly suggest that an increase in cAMP due to exposure to
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
124
VISCHER AND WOLLHEIM
IBMX and/or adenosine does not potentiate the calcium response to P2y receptor activation, ie, cAMP modulates ATPinduced vWF release at a more distal step than calcium
mobilization.
Adenosine potentiates the secretory response to thrombin.
Adenosine may potentiate the secretory response not only to
ATP but to other calcium-mobilizing agents. We therefore tested
the effect of adenosine on thrombin-induced vWF release (Fig
8). Adenosine potentiated the response to thrombin whether
added alone or together with IBMX. A significant potentiating
effect occurred at adenosine concentrations of 1 to 100 µmol/L
both alone and in the presence of IBMX, with a profile similar
to that observed for MeS-ATP (Fig 4B).
DISCUSSION
Our results demonstrate that ATP and ADP induce vWF
release from cultured HUVECs. Although the secretory response to ATP is slower than the response to thrombin, it occurs
within minutes, suggesting that vWF secretion is due to release
from preformed stores, ie, WP bodies, rather than to increased
biosynthesis and constitutive release. This conclusion is borne
out by the observation that ATP-induced vWF release consists of
high–molecular weight multimers, typical of vWF released
from WP bodies24 (Fig 2). Moreover, ATP causes a decrease in
the number of WP bodies as observed by indirect immunofluorescence (not shown).
Fig 8. Potentiating effect of adenosine on thrombin-induced vWF
release. Confluent HUVECs were incubated for 30 minutes at 37°C
with adenosine at the indicated concentrations alone (N) or together
with 100 mmol/L IBMX (W), 1 U/mL thrombin (Thr, S), or Thr/IBMX
(R). Results are the mean 6 SEM of four experiments. *P F .05 v
corresponding control without adenosine.
ATP activates P2y receptors, since it induces a rapid and
transient increase in [Ca21]i that is mimicked by the P2yspecific analog 2-methylthio-ATP. However, activation of P2y
receptors appears to be a weak stimulus for vWF release, as
indicated by the weaker secretory response to 2-methylthioATP and ADP-bS than to ATP. In addition, our results demonstrate that in vitro coactivation by adenosine is required for
ATP-induced vWF release, a potentiating effect that is mediated
by an increase in cellular cAMP content. The increase of cAMP
content in response to adenosine in conjunction with the
absence of an effect on [Ca21]i indicate that endothelial cells
express adenosine A2 receptors, in agreement with previous
reports.16,21 ATP-induced vWF release is partially inhibited by
ADA, indicating that its effect on cAMP depends on partial
conversion to adenosine in the incubation medium. The adenosine-induced cAMP formation and protein kinase A activation
are essential for the secretory response, as suggested by the
inhibitory effect of the competitive PKA inhibitor Rp-8-CPTcAMPS. Thus, adenosine, via a cAMP-dependent mechanism,
is a weak agonist for vWF release but potentiates the response
to ATP (via P2y activation). The potentiating effect of adenosine
is not restricted to ATP, since it was also observed when added
to thrombin, another well-characterized calcium-mobilizing
agent. We have previously shown that in a similar way,
epinephrine is only a weak secretagogue but a potentiator of
thrombin-induced vWF release, an effect mediated by adenylate
cyclase–coupled b-adrenergic receptors.15 Taken together, these
observations suggest that a receptor-mediated increase in cAMP
causes potentiation rather than initiation of exocytosis evoked
by calcium-mobilizing agonists.
Exocytosis from cultured endothelial cells has been associated with an increase in [Ca21]i, as observed in the secretory
response to thrombin and histamine.7,8 Incubation with the
calcium ionophore A23187 is sufficient to induce a nearmaximal secretory response.26 Further, exocytosis stimulated by
thrombin is suppressed by pretreatment with the intracellular
calcium chelator MAPTA-AM (although only weakly by removal of extracellular calcium).7 These reports strongly suggest
that exocytosis is dependent on calcium mobilization from
intracellular stores.7,14 We were therefore surprised by the weak
secretory effect of the P2y activators 2-methylthio-ATP and
ADP-bS. The maximal secretory response to 2-chloro-ATP was
stronger, but still only 55% of the thrombin response. (Even this
effect of 2-chloro-ATP may be due to partial conversion to the
potent adenosine analog 2-chloro-adenosine). The increase in
[Ca21]i in response to ATP and 2-methylthio-ATP was smaller
and shorter than the response to thrombin (Fig 7). In particular,
the second phase of elevation in [Ca21]i, known to be secondary
to calcium influx, was weak or absent in response to ATP or
2-methylthio-ATP. However, the plateau phase is thought to
play a minor role in vWF release. Removal of extracellular
calcium (by incubation with EGTA) abolishes the plateau phase
and shortens the initial [Ca21]i peak seen in response to
thrombin and superoxide anions, without a significant inhibition
of vWF release.13,14 The differences in the [Ca21]i response
therefore appear unlikely to explain the large discrepancy
between ATP (and 2-methylthio-ATP) and thrombin in terms of
vWF release. Thrombin induces only a weak cAMP response
even in the presence of IBMX. Note also that the secretory
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
ATP/ADP-INDUCED vWF SECRETION
response to either ATP or thrombin is hardly modified by IBMX
at early time points; indeed, the full potentiating effect of IBMX
is seen only by 30 minutes (Fig 3). It is therefore unlikely that
the greater initial effect of thrombin is due to coactivation of
cAMP formation. These observations suggest that in addition to
calcium and cAMP, a third, unidentified signaling pathway may
be involved in the secretory response to thrombin. A role for
coactivators of vWF release may come as a surprise, given the
well-established effect of [Ca21]i on exocytosis, in particular in
response to calcium ionophores. However, calcium ionophores
induce massive and prolonged increases in [Ca21]i that are not
achieved by receptor-mediated agonists.
cAMP is likely to be involved in the regulation of exocytosis,
judging from the potentiating effect of the cell-permeant analog
8-bromo-cAMP on vWF release induced by ATP, 2-methylthioATP, and thrombin. The actions of adenosine and epinephrine
on vWF release are associated with an increase in cellular
cAMP, and the effect of adenosine is inhibited by Rp-8-CPTcAMPS. The action of this competitive protein kinase A
inhibitor provides strong evidence that a receptor-mediated
increase in cAMP can stimulate exocytosis from cultured
endothelial cells. In contrast, an increase in platelet cAMP in
response to diverse agents such as adenosine, prostacyclin, and
even nitric oxide (via inhibition of phosphodiesterases) results
in inhibition of platelet secretion.17,27,28 From a physiologic
point of view, it is surprising that a single second messenger,
sometimes in response to the same hormone, activates endothelial cells and inhibits platelet aggregation. However, activation
of exocytosis by cAMP alone and/or in synergism with intracellular calcium should not be surprising, since it has been
observed in many secretory cell systems such as insulinsecreting b cells,29 the exocrine pancreas,30 and others. The
mechanism of this cross-talk remains poorly understood at the
molecular level, whether in endothelial cells or other secretory
cells. An effect of cAMP on calcium mobilization is highly
unlikely, since neither adenosine nor IBMX affected the calcium responses to ATP (Fig 7) or to thrombin (not shown). It is
worth emphasizing that the potentiating effect of cAMP on
calcium-mediated vWF release (in response to thrombin, ATP,
and 2-methylthio-ATP) is not restricted to adenosine, but also
occurs after stimulation with epinephrine15 and presumably
with other cAMP-raising agonists, such as prostacyclin and
other agents. This conclusion is compatible with rat hindlimb
perfusion studies showing a potentiating effect of forskolin on
vWF secretion induced by the calcium-mobilizing agents
bradykinin and platelet-activating factor.31
vWF mediates platelet aggregation, in conjunction with
fibrinogen, as well as platelet adhesion to the subendothelium.
Platelet adhesion is mediated by the interaction of vWF with the
platelet membrane protein complex GPIb-IX, which is enhanced at high shear stress.1-3 vWF is therefore considered
particularly important in the pathogenesis of arterial thrombosis. Although circulating vWF also plays a role, vWF released
from endothelial cells and trapped in the subendothelium is
particularly efficient in this process.3,32 During platelet activation, ATP and ADP are released from dense granules, where
they are stored in high concentrations (,400 mmol/L).17
Further, ATP and ADP are released from erythrocytes. This
125
release may occur even in the absence of hemolysis and is
enhanced under conditions of high shear stress.33 Our results
suggest that ATP/ADP released from activated platelets or
erythrocytes may induce vWF release from nearby endothelial
cells. Thus, extracellular ATP/ADP could facilitate the process
of thrombus formation, or at least mediate its extension from the
initial site of vascular injury, via a dual-activating effect on
platelet activation and endothelial exocytosis. Endothelial cells
express an ATP/ADPase, recently identified as CD39, which is
thought to play a major role in the regulation of thrombus
formation.21,34 This enzyme may not only protect platelets from
activation by ADP, but also control endothelial release of vWF
and surface exposure of P-selectin by regulating the balance
between ADP production and breakdown. Note that a role for
P-selectin in thrombus formation has recently been described,35
although the relative importance of endothelial and platelet
P-selectin has not yet been delineated.
Our results are at variance with an earlier study showing
potent effects of ATP, ADP, and AMP on vWF release from
cultured HUVECs at low, submicromolar concentrations.36
These low concentrations are hard to reconcile with the reported
dose-response studies for endothelial purinergic (P2y) receptors.20 The response to ADP and AMP was observed after a
30-minute lag time. This suggests that part of the response
observed may be a late effect of adenosine produced from ADP
degradation rather than a result of activation of ADP/ATP
receptors.
Exocytosis from WP bodies causes translocation of P-selectin
from the granule membrane to the cell surface.4 This surface
expression initiates leukocyte rolling and subsequent extravasation. Ischemia-reperfusion injuries are associated with tissue
infiltration by neutrophils, which is at least in part inhibited by
neutralization of P-selectin with blocking monoclonal antibodies.5,6 One group of mediators of P-selectin activation (via
exocytosis from WP bodies) are oxygen free radicals, in
particular superoxide anions generated during the reperfusion
phase.13,37 In addition, Pinsky et al38 have identified hypoxia per
se as an additional stimulus for WP body exocytosis, which
cannot be accounted for by oxygen free-radical formation. ATP
is released from the ischemic myocardium after only short
periods of coronary occlusion, ie, before actual cell necrosis.18
Adenosine is also generated during ischemia as a result of both
ATP degradation and direct release from ischemic cells.16 Our
observation that ATP, in conjunction with adenosine, induces
exocytosis from endothelial cells raises the possibility that ATP
released from ischemic tissues is a mediator of P-selectin
activation and subsequent neutrophil infiltration. At first sight, it
is unlikely that ATP is released in concentrations sufficient to
overcome degradation by endothelial ADPase. However, tissue
concentrations of ATP released from ischemic cells have not
been determined. Tissue-derived ATP production may be significantly underestimated in the content of venous effluent, since
the washout may be incomplete.18 There could also be differences in the effect of ADPase on tissue-derived (ie, basolateral)
as opposed to luminal ATP/ADP.
In cultured endothelial cells, extracellular ATP is an agonist
for endothelial exocytosis that induces vWF release and, by
inference, increases surface expression of P-selectin. ATP-
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
126
VISCHER AND WOLLHEIM
induced exocytosis may be involved in thrombus formation,
and also in ischemia-induced neutrophil infiltration and tissue
damage. The effect of ATP is mediated by dual activation of
ATP (P2y) and adenosine (A2) receptors. At the intracellular
level, an increase in both [Ca21]i and cAMP is required for
ATP-induced vWF release. cAMP potentiates the secretory
response to calcium-mobilizing agents. Future studies will
clarify whether additional agents/hormones known to activate
adenylate cyclase are also involved in the regulation of endothelial exocytosis.
ACKNOWLEDGMENT
We thank Nicole Aebischer for technical assistance, and Werner
Schlegel for insightful advice.
REFERENCES
1. Wagner DD: Cell biology of von Willebrand factor. Annu Rev
Cell Biol 6:217, 1990
2. Sadler JE: von Willebrand factor. J Biol Chem 266:22777, 1991
3. Moake JL, Turner NA, Stathopoulos NA, Nolasco LH, Hellums
JD: Involvement of large plasma von Willebrand factor multimers and
unusually large vWf forms derived from endothelial cells in shear
stress–induced platelet aggregation. J Clin Invest 78:1456, 1986
4. Mayadas TN, Johnson RC, Rayburn H, Hynes RO, Wagner DD:
Leukocyte rolling and extravasation are severely compromised in P
selectin–deficient mice. Cell 74:541, 1993
5. Weyrich AS, Ma XL, Lefer DJ, Albertine KH, Lefer AM: In vivo
neutralization of P-selectin protects feline heart and endothelium in
myocardial ischemia and reperfusion injury. J Clin Invest 91:2620,
1993
6. Winn RK, Liggitt D, Vedder NB, Paulson JC, Harlan JM:
Anti-P-selectin monoclonal antibody attenuates reperfusion injury to
the rabbit ear. J Clin Invest 92:2042, 1993
7. Birch KA, Pober JS, Zavoico GB, Means AR, Ewenstein BM:
Calcium/calmodulin transduces thrombin-stimulated secretion: Studies
in intact and minimally permeabilized human umbilical vein endothelial
cells. J Cell Biol 118:1501, 1992
8. Hamilton KK, Sims PJ: Changes in cytosolic Ca21 associated with
von Willebrand factor release in human endothelial cells exposed to
histamine. J Clin Invest 79:600, 1987
9. Ribes JA, Francis CW, Wagner DD: Fibrin induces release of von
Willebrand factor from endothelial cells. J Clin Invest 79:117, 1987
10. Hattori R, Hamilton KK, McEver RP, Sims PJ: Complement
proteins C5b-9 induce secretion of high molecular weight multimers of
endothelial von Willebrand factor and translocation of granule membrane protein GMP-140 to the cell surface. J Biol Chem 264:9053, 1989
11. Foreman KE, Vaporciyan AA, Bonish BK, Jones ML, Johnson
KJ, Glovsky MM, Eddy SM, Ward PA: C5a-induced expression of
P-selectin in endothelial cells. J Clin Invest 94:1147, 1994
12. Datta YH, Romano M, Jacobson BC, Golan DE, Serhan CN,
Ewenstein BM: Peptido-leukotrienes are potent agonists of von Willebrand factor secretion and P-selectin surface expression in human
umbilical vein endothelial cells. Circulation 92:3304, 1995
13. Vischer UM, Jornot L, Wollheim CB, Theler JM: Reactive
oxygen intermediates induce regulated secretion of von Willebrand
factor from cultured human vascular endothelial cells. Blood 85:3164,
1995
14. Birch K, Ewenstein BM, Golan DE, Pober JS: Prolonged peak
elevations in cytosolic free calcium ions, derived from intracellular
stores, correlate with the extent of thrombin-stimulated exocytosis in
single human umbilical vein endothelial cells. J Cell Physiol 160:545,
1994
15. Vischer UM, Wollheim CB: Epinephrine induces von Willebrand
factor release from cultured endothelial cells: Role of cyclic AMP in
exocytosis. Thromb Haemost 77:1182, 1997
16. Dubiak GR, El-Moatassim C: Signal transduction via P2purinergic receptors for extracellular ATP and other nucleotides. Am J
Physiol 265:C577, 1993
17. Holmsen H: Platelet secretion and energy metabolism, in Colman RW, Hirsh J, Marder VJ, Salzman EW (eds): Hemostasis and
Thrombosis: Basic Principles and Clinical Practice. Philadelphia, PA,
Lippincott, 1994, p 524
18. Forrester T: Release of ATP from the heart. Ann NY Acad Sci
603:335, 1990
19. Weiss HJ: Inherited abnormalities of platelet granules and signal
transduction, in Colman RW, Hirsh J, Marder VJ, Salzman EW (eds):
Hemostasis and Thrombosisis: Basic Principles and Clinical Practice.
Philadelphia, PA, Lippincott, 1994, p 673
20. Carter TD, Hallam TJ, Cusack NJ, Pearson JD: Regulation of
P2y-purinoceptor–mediated prostacyclin release from human endothelial cells by cytoplasmic calcium concentration. Br J Pharmacol
95:1181, 1988
21. Marcus AJ, Broekman MJ, Drosopoulos JHF, Islam N, Alyonycheva TN, Safier LB, Hajjar KA, Posnett DN, Schoenborn MA,
Schooley KA, Gayle RB, Maliszewski CR: The endothelial cell
ecto-ATPase responsible for inhibition of platelet function is CD39. J
Clin Invest 99:1351, 1997
22. Bruns RF: Adenosine receptors. Roles and pharmacology. Ann
NY Acad Sci 603:211, 1990
23. Carew MA, Paleolog EM, Pearson JD: The roles of protein
kinase C and intracellular [Ca21]i in the secretion of von Willebrand
factor from human vascular endothelial cells. Biochem J 286:631, 1992
24. Sporn LA, Marder VJ, Wagner DD: Inducible secretion of large,
biologically potent von Willebrand multimers. Cell 46:185, 1986
25. Grynkiewicz G, Poenie M, Tsien RY: A new generation of Ca21
indicators with greatly improved fluorescence properties. J Biol Chem
260:3440, 1985
26. Loesberg C, Gonsalves MD, Zandbergen J, Willems C, van Aken
WG, Stel HV, de Groot PG: The effect of calcium on the secretion of
factor VIII–related antigen by cultured human endothelial cells. Biochim Biophys Acta 763:160, 1983
27. Fisher CA, Kappa JR, Sinha AK, Cottrell ED, Reiser HJ,
Addonizio VP: Comparison of equimolar concentrations of iloprost,
prostacyclin, and prostaglandin E1 on human platelet function. J Lab
Clin Med 109:184, 1987
28. Fisch A, Michael-Hepp J, Meyer J, Darius H: Synergistic
interaction of adenylate cyclase activators and nitric oxide donor SIN-1
on platelet cyclic AMP. Eur J Pharmacol 289:455, 1995
29. Ämmälä C, Ashcroft FM, Rorsman P: Calcium-independent
potentiation of insulin release by cyclic AMP in single b-cells. Nature
363:356, 1993
30. Vajanaphanich M, Schultz C, Tsien RY, Traynor-Kaplan AE,
Pandol SJ, Barrett KE: Cross-talk between calcium and cAMPdependent intracellular signaling pathways. J Clin Invest 96:386, 1995
31. Tranquille N, Emeis JJ: The role of cyclic nucleotides in the
release of tissue-type plasminogen activator and von Willebrand factor.
Thromb Haemost 69:259, 1993
32. Baruch D, Denis C, Marteaux C, Schoevaert D, Coulombel L,
Meyer D: Role of von Willebrand factor associated to extracellular
matrices in platelet adhesion. Blood 77:519, 1991
33. Valles J, Santos MT, Aznar J, Marcus AJ, Martinez-Sales V,
Portoles M, Broekman J, Safier LB: Erythrocytes metabolically enhance collagen-induced platelet responsiveness via increased thromboxane production, adenosine diphosphate release and recruitment. J Clin
Invest 78:154, 1991
34. Marcus AJ, Safier LB, Hajjar K, Ullman HL, Islam N, Broekman
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
ATP/ADP-INDUCED vWF SECRETION
MJ, Eiroa AM: Inhibition of platelet function by an aspirin-insensitive
endothelial ADPase. J Clin Invest 88:1690, 1991
35. Subramanian M, Frenette PS, Saffaripour S, Johnson RC, Hynes
RO, Wagner DD: Defects in hemostasis in P-selectin–deficient mice.
Blood 87:1238, 1996
36. Palmer DS, Aye MT, Ganz PR, Halpenny M, Hashemi S:
Adenosine nucleotides and serotonin stimulate von Willebrand factor
release from cultured human endothelial cells. Thromb Haemost
72:132, 1994
127
37. Brown JM, Terada LS, Grosso MA, Whittmann GJ, Velasco SE,
Patt A, Harken AH, Repine JE: Xanthine oxidase produces hydrogen
peroxide which contributes to reperfusion injury of ischemic, isolated,
perfused rat hearts. J Clin Invest 81:1297, 1988
38. Pinsky DJ, Naka Y, Liao H, Oz MC, Wagner DD, Mayadas TN,
Johnson RC, Hynes RO, Heath M, Lawson CA, Stern DM: Hypoxiainduced exocytosis of endothelial cell Weibel-Palade bodies. A mechanism for rapid neutrophil recruitment after cardiac preservation. J Clin
Invest 97:493, 1996
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1998 91: 118-127
Purine Nucleotides Induce Regulated Secretion of von Willebrand Factor:
Involvement of Cytosolic Ca 2+ and Cyclic Adenosine Monophosphate−
Dependent Signaling in Endothelial Exocytosis
Ulrich M. Vischer and Claes B. Wollheim
Updated information and services can be found at:
http://www.bloodjournal.org/content/91/1/118.full.html
Articles on similar topics can be found in the following Blood collections
Hemostasis, Thrombosis, and Vascular Biology (2485 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.