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Prepublished online as a Blood First Edition Paper on January 9, 2003; DOI 10.1182/blood-2002-10-3215.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Center for Molecular and Vascular Biology and
the Laboratory of Morphology and Molecular Pathology, University of
Leuven, Belgium; and the Department of Biochemistry,
Cardiovascular Research Institute Maastricht (CARIM), University of
Maastricht, the Netherlands.
We have generated transgenic mice overexpressing the human
P2X1 ion channel in the megakaryocytic cell lineage.
Platelets from transgenic mice exhibited a gain of P2X1
ionotropic activity as determined by more prominent
P2X1-mediated Ca2+ influx and platelet shape
change. P2X1 overexpression enhanced platelet secretion and
aggregation evoked by low doses of collagen, convulxin, or the
thromboxane A2 mimetic U46619. In contrast, transgenic
platelet responses to adenosine diphosphate (ADP) or thrombin were
normal. Perfusing whole blood from transgenic mice over collagen fibers
at a shear rate of 1000 seconds Adenosine triphosphate (ATP) is released as a
cotransmitter from the sympathetic nerve endings, endothelium, and
activated platelets. It is now established that ATP and other
nucleotides act as extracellular signaling molecules.1 The
receptors that mediate the action of the adenine nucleotides belong to
2 classes, the G-protein-coupled P2Y receptors and the P2X receptors,
a family of ligand-gated ion channels.2 Seven distinct P2X
purinergic receptors have been cloned from mammalian species
(P2X1-7) and have been found to be widely expressed in
excitable and nonexcitable cells.3 Subunits of these
receptors can assemble to form homomeric and heteromeric functional
channels. All P2X receptors are cation-selective channels with almost
equal permeability to Na+ and K+ and
significant permeability to Ca2+.3 The
Ca2+ permeation through P2X receptors is considered to be
an important component of the physiologic and pathophysiologic
responses mediated by these receptors in vivo (reviewed by
Burnstock4 and North5).
In platelets, ATP and adenosine diphosphate (ADP) are present at high
concentrations in the dense granules and are coreleased during platelet
activation.6 ADP has long been recognized as an important
platelet activator, playing an essential role in enhancing secretion
and in amplifying platelet aggregation induced by other agonists.
Biologic effects of ADP are mediated by 2 distinct metabotropic
receptors, the Gq-protein-coupled P2Y1
receptor and the Gi-protein-coupled P2Y12
receptor. The latter is the target for specific antithrombotic drugs
(reviewed by Gachet7). Platelets express the
P2X1 member of the P2X family of ligand-gated ion channels,8 which mediates a rapid ATP-induced
Ca2+ influx.9,10 Because of the fast
desensitizing property of the P2X1 ion
channel11 and the lack of specific platelet
P2X1 antagonists and because platelet studies have mainly
been performed ex vivo at low extracellular Ca2+
concentrations in citrated plasma, the function of P2X1 in
platelet activation only recently started to be unraveled, and a
physiologic role of ATP in this process is now being considered.
In human platelets, the selective P2X1 agonists
To further investigate the physiologic role of the platelet
P2X1 ion channel, we have generated transgenic mice
overexpressing human P2X1 in the megakaryocytic cell
lineage. Platelets from these mice displayed a gain of P2X1
functionality accompanied by a mild prothrombotic phenotype. Combining
ex vivo and in vivo analyses of platelet function, this mouse model
enabled us to demonstrate the involvement of P2X1-mediated
Ca2+ influx and the coupled ERK2 activation in platelet
responses to collagen. We found that P2X1 overexpression
promotes platelet secretion induced by the thromboxane A2
mimetic U46619 and thereby enhances platelet aggregation. Moreover,
P2X1 overexpression also increases platelet activation and
aggregate formation under shear stress. Together, our findings suggest
a regulatory role for P2X1 during in vivo hemostasis
and thrombosis.
DNA constructs
Generation of transgenic mice
RNA isolation and RT-PCR Total RNA was extracted from mouse washed platelets and leukocytes isolated from freshly drawn citrated blood using the High Pure RNA isolation kit (Roche Diagnostics, Brussels, Belgium). During reverse transcription-polymerase chain reaction (RT-PCR), specific amplification of the human P2X1 cDNA in transgenic mouse samples was accomplished with the following primers: sense, hP2X1, 5'-GTTCCAGGAGGAGCTGGCCGCCTTCC-3'; antisense, hP2X1, 5'-GGTCTTCATGTGGGCAGCATTCAC-3'. For the specific amplification of mP2X1 cDNA, the following primers were used: sense, mP2X1, 5'-CTGCAGGATGAGCTGTCAGCCTTCTTC-3'; antisense, mP2X1, 5'-GTAGAGGCATTTCTTCATGTAGGT-3'.Materials Adenosine 5'-diphosphate (ADP),  -meATP,  -meATP,
apyrase (EC 3.6.1.5, grade 1: mixture of both high and low
ATPase/ADPase ratio isoenzymes), and the thromboxane A2
mimetic U46619 were from Sigma (St Louis, MO). ADP, -meATP, and
-meATP were purified by high-performance liquid chromatography
(HPLC) on an Adsorbosphere HS C18 7-µm, 250 × 4.6-mm
column (Alltech, Bad Segeberg, Germany) as described.12
Fibrillar collagen (Horm-type 1 collagen) was from Nycomed (Munich,
Germany) and thrombin (Dade Thrombin Reagent) was from Dade Behring
(Marburg, Germany). The MEK1/2 inhibitor U0126 was purchased from
BioMol Research Laboratories (Plymouth Meeting, MA), and D-Phe-Pro-Arg
chloromethyl ketone (PPACK) was from Calbiochem (San Diego, CA). OG
488-annexin V was from Nexins Research (Hoeven, the Netherlands).
Fura-2 acetoxymethyl ester and Pluronic F-127 came from Molecular
Probes (Leiden, the Netherlands). Recombinant saratin was produced in
the yeast Hansenula polymorpha as
described.19
Preparation of platelet-rich plasma and washed platelets Eight- to 12-week-old mice were bled under sodium pentobarbital anesthesia (6 mg/kg) from the retro-orbital plexus. Mouse blood was collected in a saline solution containing either 4 U/mL heparin, 20 µM PPACK, and 0.1 U/mL apyrase or 20 µg/mL hirudin. Platelet-rich plasma (PRP) was obtained by centrifugation at 800g for 30 seconds followed by 5 minutes at 150g. PRP from 3 animals were pooled, and the platelet counts were adjusted to 2.5 × 105 platelets/µL with autologous platelet-poor plasma (PPP). Mouse washed platelets were prepared as previously described,14 using apyrase (1 U/mL) throughout the procedure. Platelets were resuspended in Ca2+-free Tyrode buffer containing 0.35% (wt/vol) human or bovine serum albumin and 1 U/mL apyrase, at a density of 2.5 × 105 platelets/µL.Electron microscopy Platelet-rich fractions were immediately fixed overnight at 4°C in 2.5% (wt/vol) glutaraldehyde and 0.1 M phosphate buffer, pH 7.2. After centrifugation at 800g for 10 minutes, a condensed pellet of platelets was formed. After fixation in 1% OsO4 (wt/vol), 0.1 M phosphate buffer, pH 7.2, and dehydration in a graded series of ethanol, the pellets were embedded in epoxy resin. Ultrathin sections were cut and stained with uranyl acetate and lead citrate before examination with a Zeiss EM 10 electron microscope (Oberkochen, Germany).Platelet aggregation and ATP secretion analyses Light transmission during mouse platelet aggregation was recorded using apyrase-treated washed platelets in the presence of 2 mM CaCl2 on an ELVI 840 aggregometer (Elvi Logos, Milan, Italy). Shear-induced platelet aggregations were performed in an annular ring-shaped viscometer generating laminar shear (Ravenfield viscometer; Heywood, Lancashire, United Kingdom) using mouse heparinized PRP. After 3 minutes, platelet samples were collected and fixed in 1% paraformaldehyde; the percentage of platelet aggregation was calculated by comparing single platelet counts before and after shearing. ATP secretion was monitored in hirudinized PRP in parallel with platelet aggregation by adding firefly luciferase and luciferin and comparing the luminescence generated by platelet ATP release or by an ATP standard (Chrono-Lume, Kordia, The Netherlands) as previously described.14Immunoblotting Western blot detection of the human P2X1 protein in transgenic mouse platelets (8 × 108 platelets) was performed by using a polyclonal rabbit anti-hP2X1 antibody.16 Detection of ERK1/2 phosphorylation in human or mouse washed platelets (1 × 107 platelets) was accomplished with the PhosphoPlus p44/42 MAP Kinase Antibody kit (New England Biolabs, Hitchin, United Kingdom) according to the instructions of the manufacturer.Ca2+ measurements Apyrase (2 U/mL)-treated mouse washed platelets (2 × 105 platelets/µL) were loaded with 3.5 µM fura-2 acetoxymethyl ester in the presence of Pluronic F-127 for 15 minutes as described.20 The measurements were performed between 30 and 90 minutes after final platelet resuspension (0.7 × 105 platelets/µL). CaCl2 (2 mM) was added before the agonist. Fura-2 fluorescence was recorded from 0.2 mL aliquots of platelet suspension stirred at 37°C in an SLM-Aminco spectrofluorimeter (SLM Instruments, Rochester, NY) with excitation wavelengths of 340 and 380 nm and emission of 500 nm. Changes in intracellular Ca2+ concentration were monitored using the fura-2 340/380 fluorescence ratio and were calibrated according to the method of Grynkiewicz et al.21 The Ca2+ signals evoked by-meATP were assessed as the peak amplitude of
the intracellular Ca2+ rise occurring within a few
milliseconds after agonist application and returning to basal levels
after 10 seconds.
Adhesion under flow conditions Adhesion experiments under flow conditions were performed with anticoagulated mouse blood (4 U/mL heparin, 20 µM PPACK), basically as described.22 Whole blood was perfused for 4 minutes over a collagen-coated coverslip through a parallel-plate transparent flow chamber using a pulse-free pump, at a wall-shear rate of 1000 seconds 1. During the perfusion, high-resolution
microscopic transmission or fluorescent images were recorded in
real-time with a Visitech digital imaging system (Sunderland, United
Kingdom). Exposure of phosphatidylserine (PS) was detected by
postperfusion with the heparinized rinsing buffer containing
OG488-labeled annexin V (1 µg/mL). Phase-contrast and fluorescent
images were obtained from at least 10 different collagen-containing
microscopic fields that were arbitrarily chosen. When indicated,
apyrase (0.1 U/mL) was added during blood sampling; in some
experiments, blood was incubated with saratin (10 µg/mL) blocking von
Willebrand factor (VWF) binding to collagen19 1 minute
before perfusion. Area coverage from phase-contrast images was analyzed
off-line using ImagePro software (Media Cybernetics, Silver Spring,
MD). Area coverage by platelets stained with OG488-annexin V was
determined with Quanticell software (Visitech).
In vivo experiments Thromboembolism was induced by injection of a mixture of collagen (0.125 or 0.06 mg/kg) and epinephrine (60 µg/kg) into the jugular veins of anesthetized mice. When indicated, mice received 200 µg/kg U0126 1 minute before the induction of thromboembolism. For bleeding time measurements, mice were anesthetized, and 3 mm of the tail tip was amputated with a scalpel. The tail was then blotted with filter paper every 15 seconds until the paper was no longer blood stained.Statistical analyses Statistical analyses of the data were made using the nonpaired Student t test and the 2-tailed Tukey-Kramer multiple comparisons test. Survival data were analyzed using 2 × 2 contingency tables.
Generation of transgenic mice overexpressing the human P2X1 ion channel in the megakaryocytic cell lineage On zygote injection of a constructed GPIIb-hP2X1 transgene, among 19 offspring mice obtained, 4 animals were found to be transgenic by PCR screening. All founders transmitted the transgene in a Mendelian fashion. RT-PCR analyses using primer pairs that selectively amplify hP2X1 versus mP2X1 mRNAs revealed the presence of hP2X1 transcripts in the platelets of the transgenic (TG) mice (Figure 1A). The expression of the endogenous platelet mP2X1 remained comparable to that of wild-type (WT) platelets (Figure 1A). No transgene expression was found in the leukocytes (Figure 1A). Overexpression of the hP2X1 protein in platelets was demonstrated by immunoblotting of heterozygous (TG+/) and homozygous (TG+/+) mouse platelet
extracts (Figure 1B); similarly, immunohistochemistry of bone marrow
sections revealed increased P2X1 staining in the TG
megakaryocyte membranes (not shown). The homozygous mice of 2 founder
lines (denominated TG16 and TG18)
overexpressing similar amounts of platelet hP2X1 were
characterized and showed identical phenotypes. These mice had no
apparent physiologic abnormalities and displayed normal development,
survival, and reproduction. Platelet count (Table
1) and morphology (not shown), as well as
other hematologic parameters, were identical to those of WT mice, with
the exception of mild leukocytosis (Table 1). Values are represented as
mean ± SD (P = .003).
Overexpression of hP2X1 results in a gain of P2X1 functionality in the transgenic platelets Apyrase, by degrading ATP spontaneously released during blood sample handling, is needed ex vivo to protect platelet P2X1 channels from artificial desensitization.12,13 In the presence of this ectonucleotidase and physiologic Ca2+ concentrations (2 mM CaCl2), the nonhydrolyzable P2X1 selective agonists-meATP (1 µM, defined to be
the optimal concentration) (Figure 2) and
-meATP (not shown) evoked a rapid Ca2+ influx in
mouse platelets. The peak value of this intracellular Ca2+
rise was increased by approximately 50% in TG platelets compared with
WT platelets (TG, 146.5 ± 23.6 nM, n = 8; WT, 93.1 ± 21.4 nM,
n = 8; P = .0003) (Figure 2). In the same experimental
conditions, the WT and TG platelets displayed identical ADP-induced
Ca2+ increases, reflecting P2Y1-mediated
Ca2+ responses (WT, 399 ± 66 nM, n = 5; TG,
478 ± 74 nM, n = 5; P = .112).
In human platelets, P2X1 stimulation causes
Ca2+ influx and subsequent platelet shape
change.12,13 Similarly, Enhanced platelet aggregation induced by collagen in hP2X1 transgenic mice: role of the ERK2 signaling pathway In an aggregometer, the P2X1 selective agonists-meATP and -meATP did not cause platelet aggregation or
secretion either in WT or in TG mice. Functional studies of
apyrase-treated platelets from TG mice, where P2X1
desensitization is prevented, yet demonstrated strongly enhanced
platelet aggregation evoked by low and intermediate doses of collagen
(1 to 2 µg/mL) compared with WT platelets (Figure 3A). A similar increase of TG platelet
aggregation was observed after platelet stimulation with low
concentrations of convulxin, a glycoprotein VI (GPVI)-selective
agonist (0.025 to 0.04 µg/mL) (Figure 3A, right panel), indicating
that P2X1 overexpression enhances platelet aggregation
mediated by the collagen receptor GPVI. Platelet aggregations induced
by higher collagen (4 µg/mL or greater) or convulxin (0.05 µg/mL or
greater) concentrations were identical for WT and TG platelets
(not shown).
Threshold concentrations of collagen, which only caused shape change of
WT platelets, produced full aggregation of TG platelets (Figure 3A).
Consistent with an event requiring functional P2X1 channels, the enhanced reactivity to collagen was abrogated when the
apyrase treatment was omitted (Figure 3B) or when these platelets were
pretreated with Further analyses of TG platelet aggregation revealed an increased
response to low concentrations of the thromboxane A2
mimetic U46619 (less than 2 µM) (Figure
4A). In contrast, TG platelet aggregation
provoked by any concentration of HPLC-purified ADP or of thrombin
occurred normally (Figure 4B).
Enhanced platelet secretion induced by collagen and U46619 in hP2X1 transgenic mice To investigate the mechanism responsible for the observed increased aggregation of TG platelets, we compared platelet secretion induced by low concentrations of collagen or of U46619 in WT and TG PRP. As in human platelets, the desensitization of P2X1 with-meATP strongly inhibited ATP secretion from WT platelets,
indicating a P2X1 contribution to this platelet response
(not shown). Figure 5 shows increased ATP
secretion of TG platelets (1.9 ± 0.6- and 3.3 ± 0.2-fold
increases in response to 0.5 µg/mL collagen and 4 µM U46619,
respectively [P < .001; n = 3]) paralleled with
enhanced platelet aggregation. Thus, P2X1 overexpression in
TG platelets potentiates platelet-dense granule release initiated by
low doses of collagen or of the thromboxane A2 mimetic
U46619, leading to the amplification of the initial platelet response
and the completion of aggregation. ATP secretion triggered by ADP or
thrombin was found to be normal (not shown).
Increased whole blood aggregate formation and platelet phosphatidylserine exposure on a collagen-coated surface under flow To further investigate the TG platelet hyperreactivity to collagen, aggregate formation and exposure of coagulation-active negatively charged phosphatidylserine (PS) on a collagen surface were analyzed in whole mouse blood under conditions of flow. Using videomicroscopy, aggregate formation was monitored from phase-contrast images, and PS exposure (procoagulant activity) was imaged after annexin V staining. As previously described,22 when WT mouse blood was perfused over collagen at a shear rate of 1000 seconds1, platelets tethered, adhered, and assembled on
the surface, with the adherent cells responding by a rapid increase in
cytosolic Ca2+,22 and subsequent
surface-exposure of PS (Figure 6). A
similar picture was observed following perfusion of TG blood (Figure
6). The use of ATP/ADP-degrading apyrase, needed to protect
P2X1 from ATP-provoked desensitization, considerably
reduced aggregate formation and platelet PS exposure after the
perfusion of WT blood (Figure 6A-B). This is compatible with the
reported role of the P2Y1 and P2Y12 receptors
interacting with released ADP during aggregate formation on
collagen.23,24 Perfusion of apyrase-treated TG blood yet
resulted in prominent platelet aggregation coinciding with high PS
exposure (Figure 6A-B), suggesting that P2X1 overexpression in TG platelets has compensated the inhibitory effects of apyrase by
promoting ATP-dependent platelet adhesion, activation, and aggregation
under these flow conditions.
Figure 6 also shows how aggregate formation and PS exposure by
apyrase-treated TG platelets were abrogated by saratin (Figure 6A-B),
which, by blocking VWF binding to collagen, prevents
GPIb Interestingly, area coverage by platelet aggregates and PS exposure was reduced by 30% to 35% during the perfusion of apyrase-treated TG blood in the presence of U0126 (Figure 6C-D). These data indicate the involvement of the ERK2 pathway in platelet aggregate formation on collagen under flow. U0126 did not affect aggregate formation or PS exposure when WT blood was perfused in P2X1 nonprotective conditions (no apyrase) (not shown). These observations thus support the existence of a P2X1-ERK2 pathway contributing to platelet activation by collagen and surface-bound VWF. Potent shear-induced aggregation of TG platelets Platelet aggregation induced by shear stress can be measured in a viscometer producing a laminar flow. At high shear stress, VWF binding to GPIb is essential to induce
IIb3-dependent platelet aggregation.25 We have investigated whether
P2X1 would contribute to shear-dependent platelet
aggregation. For this purpose, platelet aggregations were performed in
apyrase-treated heparinized PRP from WT and TG mice, at a shear rate of
9000 seconds1 corresponding to a shear stress of 124 dyne/cm2. At this shear rate, the transgenic platelets
underwent potent aggregation (52.4% ± 15.5% of aggregation)
compared with negligible aggregation of WT platelets (9.9% ± 6.3%)
(Figure 7), showing that the TG platelets
exhibited increased ability to respond to shear stress.
Increased thrombotic tendency in transgenic mice Bleeding times, considered to reflect primary hemostasis in vivo, were identical for WT and TG mice (WT, 5.07 ± 2.05 minutes, n = 22; TG, 4.98 ± 2.41 minutes, n = 12). Because in vitro collagen-induced transgenic platelet activation and aggregation were greatly enhanced, we used an in vivo model of pulmonary thromboembolism by intravenous injection of a mixture of a low dose of collagen (0.06 mg/kg body weight) and epinephrine (60 µg/kg). As shown in Figure 8A, 80% of the TG mice died after 4 minutes compared with only a 30% mortality rate for WT mice. These results indicate that P2X1 overexpression generates a prothrombotic phenotype.
Antithrombotic protection by blockade of the ERK2 pathway To further examine the importance of the P2X1-coupled ERK2 signaling pathway for platelet function in vivo, we analyzed the effect of a 1-minute pretreatment with U0126 (200 µg/kg) on the mortality of TG mice. All mice pretreated with U0126 survived the injection of collagen (0.06 mg/kg) plus epinephrine (60 µg/kg) (Figure 8A). Inhibition of ERK2 also conferred antithrombotic protection to WT mice injected with a mixture of a higher dose of collagen (0.125 mg/kg) plus epinephrine (60 µg/kg); the number of survivors reached 90% compared with 40% in controls (Figure 8B).
We have generated transgenic mice overexpressing the human
ATP-gated P2X1 ion channel in the megakaryocytic cell
lineage. Platelets from these mice displayed a gain of P2X1
ionotropic activity, as shown by more prominent Ca2+ influx
and platelet shape change triggered by the 2 P2X1-selective agonists, Enhanced secretion and aggregation of the transgenic platelets in response to collagen corroborate our previous findings in healthy human platelets.13,14 We have reported that low concentrations of collagen cause early minor ATP release that elicits a rapid P2X1-mediated Ca2+ influx, contributing to enhancement of the platelet release reaction, thus completing platelet aggregation. It is therefore likely that the enhanced TG platelet response to collagen results from an increased number of functional ATP-responsive P2X1 channels expressed at the surfaces of TG platelets. The higher level of P2X1 activation thereby leads to enhanced platelet secretion and aggregation. Similarly, the increased TG platelet aggregation induced by low concentrations of the thromboxane A2 mimetic U46619 was related to enhanced platelet secretion induced by this agonist. This is in agreement with earlier studies on the importance of secreted products, mainly ADP, during U46619-induced platelet aggregation.26 Collagen activates platelets by transducing signals through glycoprotein VI (GPVI). We investigated whether the observed enhanced platelet aggregation induced by collagen depended on this glycoprotein by using the GPVI-selective agonist convulxin. We found that TG platelet aggregation induced by low concentrations of convulxin was similarly enhanced, as with collagen, suggesting cooperation between secreted ATP- and GPVI-mediated signaling under mild stimulation of this receptor. In agreement with this finding and using ADP receptor antagonists, Quinton et al27 have reported a role for secreted ADP during platelet aggregation provoked by low concentrations of convulxin, whereas platelet aggregation at higher concentrations of convulxin was unaffected by these agents. In human platelets, we have identified the ERK2 signaling pathway as an intracellular mechanism subserving the function of the ATP-gated P2X1 ion channel during platelet aggregation induced by low concentrations of collagen. We have shown that the P2X1-mediated ERK2 activation is needed to amplify dense-granule release initiated by this agonist. As in human platelets, the aggregation of wild-type mouse platelets evoked by low concentrations of collagen was abolished after blockade of the ERK2 pathway with the selective MEK1/2 inhibitor U0126. Accordingly, the increased collagen-induced aggregation of transgenic platelets coincided with the up-regulation of ERK2 and could equally be abolished by U0126. It appears that P2X1 and ERK2 are 2 components of a common signaling cascade in human and mouse platelets. Thus, even though the P2X1-mediated Ca2+ influx seems to be small, this necessarily local signal close to the plasma membrane, where ERK2 is also translocated, is likely to generate a significant local trigger for the promotion of Ca2+- and ERK2-dependent secretion. To investigate the physiologic relevance of the
P2X1-coupled ERK2 pathway in thrombosis, we used an in vivo
model of pulmonary thromboembolism by injection of a mixture of
collagen and epinephrine. We showed that inhibition of the ERK2 pathway
by U0126 fully protected transgenic mice against lethal thrombosis
induced by a low dose of collagen plus epinephrine. Wild-type mice were
also partly protected after the administration of a higher dose of
collagen. Together with the observation that
P2X1-overexpressing transgenic platelets were hyperreactive
to collagen in an ERK2-dependent fashion, our in vivo data support the
existence of a P2X1-ERK2 signaling axis involved Shear-induced platelet aggregation requires initial VWF binding to
platelet GPIb Where rapid blood flow creates high wall shear rates, such as in
arterioles in the healthy circulation or in atherosclerotic arteries
with restricted lumen, platelet thrombus formation depends on VWF
immobilized on extracellular matrix components, in particular collagens.30 Compatible with the enhanced reactivity of
transgenic platelets to collagen and shear stress, perfusion of whole
blood from transgenic mice over a collagen surface at a shear rate of 1000 seconds In vitro, because of the rapid desensitization of P2X1 by
spontaneously released ATP, a high concentration of apyrase is often required to demonstrate this ion channel function.12-14 In
addition, in the present experiments with isolated mouse platelets,
apyrase was needed to detect P2X1 knock-out mice display male infertility resulting from reduced neurogenic vas deferens contraction.34 In agreement with our study, preliminary data recently presented indicate impaired in vitro platelet aggregation induced by low doses of collagen.35 Furthermore, the perfusion of whole blood from these mice over a collagen surface revealed reduced aggregate formation under flow conditions.35 Finally, because P2X1 overexpression in platelets generates a prothrombotic tendency, we can speculate that pathologic deregulation of P2X1 expression may have a significant impact on platelet activation and may contribute to abnormal thrombosis. It is noteworthy that, in other tissues, the up-regulation of P2X1 mRNA levels has been described in pathophysiologic conditions (reviewed by Burnstock4). Thus, P2X1, as the predominant P2 receptor subtype in bladder smooth muscle, showed a considerably increased expression in the symptomatically obstructed bladder. The up-regulation of P2X1 mRNA in the hearts of rats with congestive heart failure has been reported. Overall, the present study provides evidence that the P2X1 ion channel plays a role in mediating the biologic effects of ATP during platelet activation. Platelet overexpression of P2X1 resulted in increased platelet secretion and aggregation triggered by collagen and thromboxane A2 but also enhanced platelet responses under shear stress. A novel physiologic role of the P2X1-ERK2 signaling pathway in hemostasis and thrombosis is also proposed.
Submitted October 24, 2002; accepted January 4, 2003.
Prepublished online as Blood First Edition Paper, January 9, 2003; DOI 10.1182/blood-2002-10-3215.
Supported by the bilateral scientific and technological cooperation between Flanders and Hungary (BIL00/12) and from Fonds voor Wetenschappelijk Onderzoek (FWO) project G.0227.03. C.O. is holder of a postdoctoral research mandate of the FWO. E.T.Z. is recipient of a doctoral KULeuven scholarship. J.V. is holder of the Aventis Chair of Hemostasis Research.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Marc Hoylaerts, Center for Molecular and Vascular Biology, University of Leuven, Herestraat 49, B-3000 Leuven, Belgium; e-mail: marc.hoylaerts{at}med.kuleuven.ac.be.
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  M. Mazzucato, M. R. Cozzi, M. Battiston, M. Jandrot-Perrus, M. Mongiat, P. Marchese, T. J. Kunicki, Z. M. Ruggeri, and L. De Marco Distinct spatio-temporal Ca2+ signaling elicited by integrin {alpha}2{beta}1 and glycoprotein VI under flow Blood, September 24, 2009; 114(13): 2793 - 2801. [Abstract] [Full Text] [PDF]
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A. Bonnefoy, K. Daenens, H. B. Feys, R. De Vos, P. Vandervoort, J. Vermylen, J. Lawler, and M. F. Hoylaerts Thrombospondin-1 controls vascular platelet recruitment and thrombus adherence in mice by protecting (sub)endothelial VWF from cleavage by ADAMTS13 Blood, February 1, 2006; 107(3): 955 - 964. [Abstract] [Full Text] [PDF]
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C. Oury, E. Sticker, H. Cornelissen, R. De Vos, J. Vermylen, and M. F. Hoylaerts ATP Augments von Willebrand Factor-dependent Shear-induced Platelet Aggregation through Ca2+-Calmodulin and Myosin Light Chain Kinase Activation J. Biol. Chem., June 18, 2004; 279(25): 26266 - 26273. [Abstract] [Full Text] [PDF]
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P. R.-M. Siljander, I. C. A. Munnix, P. A. Smethurst, H. Deckmyn, T. Lindhout, W. H. Ouwehand, R. W. Farndale, and J. W. M. Heemskerk Platelet receptor interplay regulates collagen-induced thrombus formation in flowing human blood Blood, February 15, 2004; 103(4): 1333 - 1341. [Abstract] [Full Text] [PDF]
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 J. A. Calvert and R. J. Evans Heterogeneity of P2X Receptors in Sympathetic Neurons: Contribution of Neuronal P2X1 Receptors Revealed Using Knockout Mice Mol. Pharmacol., January 1, 2004; 65(1): 139 - 148. [Abstract] [Full Text] [PDF]
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E. Toth-Zsamboki, C. Oury, H. Cornelissen, R. De Vos, J. Vermylen, and M. F Hoylaerts P2X1-mediated ERK2 Activation Amplifies the Collagen-induced Platelet Secretion by Enhancing Myosin Light Chain Kinase Activation J. Biol. Chem., November 21, 2003; 278(47): 46661 - 46667. [Abstract] [Full Text] [PDF]
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C. Vial, S. J. Pitt, J. Roberts, M. G. Rolf, M. P. Mahaut-Smith, and R. J. Evans Lack of evidence for functional ADP-activated human P2X1 receptors supports a role for ATP during hemostasis and thrombosis Blood, November 15, 2003; 102(10): 3646 - 3651. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
508 relative to the
initiation start site (TOPO TA cloning kit; Invitrogen, Carlsbad, CA)
was performed using the following primers: sense,
5'-AGGAAGTGGGTAAATGTCCTACTC-3'; antisense,
5'-TCCCAAACGTCCTAAACAGGAATGG-3'. The
XhoI-HindIII promoter fragment was excised from
the mGPIIb-PCR2.1-TOPO plasmid and inserted into the pGL3-basic
luciferase reporter vector (Promega, Leiden, the Netherlands) digested
with XhoI and HindIII. Megakaryocytic human
erythroblastic leukemia (HEL) and nonmegakaryocytic HeLa cell lines
were transiently transfected with the resultant reporter construct, and
megakaryocytic-specific promoter activity was verified as
described
) represent the surface
area coverage by platelets calculated from the phase-contrast images at
the end of the perfusion period. White bars (
) represent the area
coverage by platelets binding OG488-annexin V after perfusion
(platelets exposing negatively charged PS) (n = 3-6)
(*P < .001 vs WT;
#P < .001 vs WT + apyrase;
¶P < .001 vs TG + apyrase;
among
several other components