From the Medizinische Klinik IV,
Universitätsklinikum Benjamin Franklin, Freie Universität
Berlin, Berlin 12200, the § Medizinische
Universitätspoliklinik, Bonn D-53111, and the ¶ Institut
für Neurophysiologie, Universität zu Köln,
Köln D-50931, Germany
Received for publication, October 18, 2000, and in revised form, December 13, 2000
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ABSTRACT |
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Dinucleoside diphosphates,
Ap2A, Ap2G, and Gp2G
represent a new class of growth-promoting extracellular mediators,
which are released from granules after activation of platelets. The
presence of theses substances was shown after purification from a
platelet concentrate. The substances were identified by UV
spectrometry, retention time comparison with authentic substances,
matrix-assisted laser desorption/ionization mass spectrometry,
post-source-decay matrix-assisted laser desorption/ionization mass
spectrometry, and enzymatic analysis. Ap2A,
Ap2G, and Gp2G have growth-stimulating effects
on vascular smooth muscle cells in nanomolar concentrations as shown by
[3H]thymidine incorporation measurements. The calculated
EC50 (log M; mean ± S.E.) values
were To further evaluate the pathogenesis of hypertension, there is a
continued interest in the identification of novel endogenous compounds
with growth-stimulating effects on vascular smooth muscle cells
(VSMCs).1 In this context,
novel endogenous nucleotides have been recognized as powerful
vasoactive messengers.
In the last decade dinucleoside polyphosphates received
considerable attention in view of their multiple biological and
pharmacological activities. Dinucleoside polyphosphates were identified
in prokaryotic (1), eukaryotic, and mammalian cells (2).
Di(adenosine-5') tri- and tetraphosphates (Ap3A,
Ap4A) were the first dinucleoside polyphosphates to be
identified in human platelets (3, 4). Di(adenosine-5') pentaphosphate
(Ap5A) and di(adenosine-5') hexaphosphate (Ap6A) have been described as vasoconstrictive substances
isolated from human platelets (5). Recently, di(adenosine-5')
heptaphosphate (Ap7A) has been isolated in human platelets
and has been postulated to play a role in the control of vascular tone
(6). Furthermore, dinucleoside polyphosphates containing adenosine and
guanosine (adenosine-5' oligophospho 5'-guanosines (ApnG;
n = 3-6)) or containing two guanosines (guanosine-5'
oligophospho 5'-guanosines (GpnG; n = 3-6))
were identified in human platelets (7). ApnG (n = 5-6) have a vasoconstrictive effect, whereas GpnG do not
affect vascular tone in the isolated perfused rat kidney. Both
ApnGs and GpnGs (with n = 3-6) are
growth-stimulating factors of VSMCs. (7). Several purine receptor
subtypes (P2 receptors) mediating the actions of dinucleoside polyphosphates have been established with different physiological effects (8, 9). The P2 receptors are divided into two families of
ligand-gated ion channel and G protein-coupled receptors termed P2X and
P2Y receptor, respectively. There are eight mammalian P2X receptors
(P2X1-8) (10, 11) and five mammalian P2Y receptors
(P2Y1, P2Y2, P2Y4,
P2Y6, and P2Y11) that have been cloned (9).
From these results the question arose as to whether
P1,P2-dinucleoside diphosphates containing two
adenosine or guanosine groups or an adenosine and a guanosine group
also occur in humans. There is one report on the existence of
diadenosine diphosphate (Ap2A) isolated from human cardiac
tissue (12). In contrast to Ap2A the
P1,P2-dinucleoside diphosphates
Ap2G and Gp2G have not been described as
endogenous substances so far in the literature.
Here the existence of diadenosine diphosphate (Ap2A),
adenosine guanosine diphosphate (Ap2G), as well as
diguanosine diphosphate (Gp2G) in releasable granules of
human platelets is shown for the first time and their
growth-stimulating effect on cultured vascular smooth muscle cells is described.
Chemicals
HPLC water (gradient grade) and acetonitrile were from Merck
(Germany). All other substances were purchased from Sigma (Germany).
Purification of Dinucleoside Diphosphates from Human
Platelets
Dinucleoside diphosphates were isolated from human platelets
unsuitable for transfusion. The platelets were suspended in an isotonic
salt solution and centrifuged at 2500 × g for 5 min. The pellet was resuspended in an isotonic salt solution and centrifuged again (2500 × g, 5 min). The supernatant was
aspirated, and the platelet pellet was frozen to Chromatography
Preparative Reversed Phase Chromatography--
Triethylammonium
acetate (TEAA) was added to the supernatant (final TEAA concentration
40 mM), and hydrophobic substances were concentrated on a
C18 reversed phase column (Lichroprep, 310 × 65 mm, 40-65 µm,
Merck, Germany) using 40 mM TEAA in water (eluent A; flow,
2 ml/min). After removing substances not binding to the column with
aqueous 40 mM TEAA (flow: 2 ml/min), the adsorbed substances were eluted with 20% acetonitrile in water (eluent B). The
elution was detected by measuring the UV absorption at 254 nm. The
eluate was lyophilized and stored frozen at Affinity Chromatography--
The lyophilized eluate of the
reversed phase chromatography was dissolved in 1 M ammonium
acetate (eluent C; pH 9.5) and purified further with affinity
chromatography (step 4). The affinity chromatography gel, phenyl
boronic acid coupled to a cation-exchange resin (BioRex 70, Bio-Rad),
was synthesized according to Barnes (13). The affinity resin was packed
into a glass column (150 × 20 mm) and equilibrated with 1 M NH4Ac (pH 9.5; flow, 2 ml/min). The pH of the
eluate from the reversed phase column was adjusted to pH 9.5 and loaded
to the affinity column. The column was washed with 1 M
NH4Ac (pH 9.5) with a flow rate of 2 ml/min. Binding
substances were eluted with 1 mM HCl (eluent D). Fractions
were monitored with a UV detector at 254 nm. The eluate was frozen and lyophilized.
Reversed Phase Chromatography--
The eluate of the affinity
chromatography was desalted by reversed phase chromatography (step 4).
The reversed phase column (Supersphere, 210 × 4.1 mm, 4 µm,
Merck) was equilibrated with aqueous 40 mM TEAA solution
(eluent A). The sample, with 40 mM TEAA in water added, was
pumped at a rate of 0.5 ml/min onto the column. After washing the
column with 15 ml of eluent A, the fraction of interest was eluted with
35% acetonitrile in water (eluent E). The resulting fractions were
lyophilized and stored at Analytical and Anion-exchange Chromatography--
The
eluate of the reversed phase column was lyophilized, dissolved
in 0.5 ml of 20 mM K2HPO4 in water
(pH 8.0, eluent F), and chromatographed by using an
anion-exchanger (DEAE 5PW, 150 × 20 mm, 10 µm, Tosohaas, Japan)
using 20 mM K2HPO4 in water, pH 8.0 (eluent F), and 20 mM K2HPO4 and 1 M NaCl (pH 8.0) (eluent G) in water using the following
gradient: 0-10 min: 0-5% G; 10-105 min: 5-35% G; 105-110 min:
35-100% G; 110-120 min: 100% G; 120-121 min: 100-0% G. The flow
rate was 2.0 ml/min, and absorption was measured at 254 nm (step 6).
Reversed Phase Chromatography--
Thereafter, each fraction of
the anion-exchange chromatography with a significant UV absorbance at
254 nm was chromatographed on an analytical reversed phase column
(Supersphere RP C18 end-capped, 250 × 4.6 mm, Merck) using 40 mM TEAA in water (eluent A) and 100% acetonitrile (eluent
H) with the following gradient: 0-4 min: 0-4% H; 4-64 min: 4-11%
H; 64-70 min: 11-70% H (flow, 0.5 ml/min; step 7). The resulting
fractions were lyophilized and stored at Identification of the P1,P2-dinucleoside
Diphosphates by Reversed Phase Chromatography
To test the fractions for homogeneity, a small part
(1/1000) of the desalted and lyophilized fractions of the
anion-exchange chromatography were chromatographed on a second reversed
phase HPLC column (Poros, R 2/H, 2.1 × 100 mm, Perseptive
Biosystems). The column was run in the gradient mode (flow rate, 300 µl/min) with 10 mM K2HPO4 and 2 mM tetrabutyl-ammonium hydrogen sulfate in water (eluent I)
and 80% acetonitrile in water (eluent J; gradients: 0-30.5 min,
0-30% J; 30.5-31 min, 30-50% J; 31-34.5 min, 50% J; 34.5-35
min, 0% J). The elution was detected by measuring the UV absorption at
254 nm.
Matrix Assisted Laser Desorption/Ionization Mass Spectrometry
The desalted and lyophilized fractions of the anion-exchange
chromatography were examined by matrix-assisted laser
desorption/ionization mass spectrometry (MALDI-MS) (14). A
reflectron-type time-of-flight mass spectrometer (Reflex III,
Bruker-Franzen, Germany) was used according to Hillenkamp (14). The
sample was mounted on an x, y, z movable stage allowing for irradiation
of selected sample areas. In this study, a nitrogen laser (Laser
Science Inc.) with an emission wavelength of 337 nm and 3-ns pulse
duration was used. The laser beam was focused to a diameter typically
of 50 µm at an angle of 45° to the surface of the target.
Microscopic sample observation was possible via a diachronic mirror in
the beam path. 10-20 single spectra were accumulated for a better
signal-to-noise ratio. The concentrations of the analyzed substances
were 1-10 µM in double-distilled water. 1 µl of the
analyte solution was mixed with 1 µl of the matrix solution. For this
study, a solution of 50 mg/ml 3-hydroxypicolinic acid was used. For
calibration of the mass spectra, diadenosine hexaphosphate
(Ap6A) was used as external standard. The mixture was
gently dried on an inert metal surface before introduction into the
mass spectrometer. The mass accuracy was in the range of ~0.05%.
UV Spectroscopy
The desalted, lyophilized fractions of the reversed phase
chromatography (step 8) were dissolved in water (100 µl). To measure UV spectra at different pH the pH values of the solutions were adjusted
to 3.0, 7.0, and 9.0 by 0.1 M HCl and 0.1 M
NaOH, respectively. The UV absorbance of the fractions were determined
by a UV-visible spectrometer (DU-600, Beckman) at wavelengths between
190 and 400 nm with a scan speed of 400 nm/min.
Platelet Activation by Thrombin and Purification of
Dinucleoside Diphosphates AP2A, AP2G,
GP2G, and Serotonin from the Supernatant
Three platelet concentrates (each 200 ml; 107
platelets/µl) were resuspended in 600 ml of a buffer containing 0.14 M NaCl, 0.15 mM Tris-HCl. To prevent premature
activation 0.35% (w/v) albumin was added to the buffer. The
resuspended platelet concentrates were divided into three parts.
To test the release of the dinucleoside diphosphates, one aliquot was
incubated with thrombin (0.05 units/ml) for 1 min. Preliminary experiments showed that fibrinogen binding in platelets did not exceed
2000 molecules/cell. After stimulation with thrombin, the fibrinogen
binding rose 20- to 30-fold. Determination of fibrinogen was performed
exactly as described previously (15). Moreover, the concentration of
serotonin was determined in the supernatant. As control the second
aliquot was not incubated with thrombin.
For purification of dinucleoside diphosphates Ap2A,
Ap2G, and Gp2G from the supernatant, platelets
were removed by centrifugation (4000 rpm, 4 °C, 10 min). The
supernatant was deproteinated with 0.6 M (final
concentration) perchloric acid and centrifuged (4000 rpm, 4 °C, 5 min). After adjusting the pH to 7.0 with 5 M KOH the
precipitated proteins and KClO4 were removed by
centrifugation (4000 rpm, 4 °C, 5 min). The supernatants of both
aliquots of the platelet concentrates were chromatographed according to
chromatographic steps for the purification of dinucleoside diphosphates
from platelets. Dinucleoside diphosphates Ap2A,
Ap2G, and Gp2G were identified by retention
time comparison with authentic substances as well as MALDI-MS.
For the measurement of the total endogenous serotonin content, a method
described by Hervig et al. (16) was used. Briefly, 600 µl of the platelet concentrate as prepared above was mixed with
200 µl of a 2.8 M perchloric acid solution containing
dithiothreitol (40 mM) to precipitate the proteins. The
precipitate was removed by centrifugation (8000 × g, 2 min), and 520 µl of the supernatant was neutralized with 130 µl of
3 M K2HPO4. The precipitated
potassium perchlorate was removed by a second centrifugation (8000 × g, 2 min).
The supernatant was transferred and was directly analyzed by the
reversed phase chromatographic method of Anderson et al. (17). 100 µl of the supernatant was injected onto a reversed phase
column (Supersphere, 210 × 4.1 mm, 4 µm, Merck) eluted with 0.1 M phosphate buffer (pH 4.5) containing 250 µl/liter
triethylamine, 150 mg/liter sodium octylsulfate, and 20% (v/v)
methanol (flow rate, 0.5 ml/min). The fluorescence was detected using
an SP920 intelligent fluorescence detector (Jasco) with excitation and emission wavelength settings of 285 and 350 nm, respectively. Quantification of serotonin was done by using a calibration curve.
For the measurement of the released serotonin after thrombin
stimulation, a method described by Hervig et al. (16) was
used. Briefly, 600 µl of the platelet concentrate as prepared above were incubated with 10 NIH units of thrombin (10 µl) for 10 min. After removing the platelet remnant by centrifugation (8000 × g, 30 s), 450 µl of supernatant was mixed with 150 µl of the perchloric acid/dithiothreitol solution and centrifuged
again (8000 × g, 2 min). 400 µl of the supernatant
was neutralized with 100 µl of a 3 M solution of
K2HPO4, recentrifuged as above, and injected to
the chromatography using the method described by Anderson et al. (17).
Synthesis and Chromatography of Authentic
P1,P2-dinucleoside Diphosphates
In contrast to diadenosine diphosphate and diguanosine
diphosphate, adenosine guanosine diphosphate was commercially not
available. Therefore, synthesis of adenosine guanosine diphosphate was
necessary to control the authenticity of the isolated substances.
Adenosine guanosine diphosphate was synthesized and chromatographed
following a study described elsewhere (18). Briefly, Ap2G
was synthesized by mixing AMP (25 mM) and GMP (25 mM) as substrates in the presence of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (2.5 M),
HEPES (2 M), and magnesium chloride (125 mM).
The substances were dissolved in water, thoroughly mixed with a vortex
mixer, and incubated at 37 °C at pH 6.5 for 48 h. The reaction
mixture was concentrated on a preparative C18 reversed phase column
(condition described above). The concentrate was displaced on a
reversed phase column (carrier: 40 mM TEAA in water (eluent
A); displacer: 160 mM n-butanol (eluent K), flow
100 µl/min). As a result of displacement chromatography, anion-exchange chromatography yielded baseline separated dinucleoside diphosphates (Ap2A, Ap2G, and
Gp2G).
Commercially available diadenosine diphosphate and diguanosine
diphosphate are contaminated with mononucleotides. Therefore, these
P1,P2-dinucleoside diphosphates were purified
by displacement chromatography using a reversed phase column
(conditions as describe above) before testing the authenticity of the
isolated substances.
Enzymatic Cleavage Experiments
Aliquots of the fractions containing homogenous nucleotides from
the reversed phase chromatography (steps 7 and 8 of the purification procedure), were incubated with enzymes as described in the following. The samples were dissolved: (a) in 20 µl 200 mM Tris buffer (pH 8.9) and incubated with 5'-nucleotide
hydrolase (3 milliunits (mU); from Crotalus durissus, EC
3.1.15.1, from Roche Molecular Biochemicals, Germany, purified
according to Sulkowski and Laskowski (19) for 9 min at 37 °C;
(b) in 20 µl of 200 mM Tris and 20 mM EDTA buffer (pH 7.4) and incubated with 3'-nucleotide
hydrolase (1 mU; from calf spleen, EC 3.1.16.1, from Roche Molecular
Biochemicals, Germany) for 1 h at 37 °C; and (c) in
20 µl of 10 mM Tris, 1 mM ZnCl2,
and 1 mM MgCl2 buffer (pH 8.0) and incubated
with alkaline phosphatase (1 mU; EC 3.1.3.1, from calf intestinal
mucosa, from Roche Molecular Biochemicals, Germany) for 1 h at
37 °C. The reaction was terminated by an ultrafiltration with a
centrifuge filter (exclusion limit, 10 kDa). After filtration of the
enzymatic cleavage products, the filtrate, dissolved in 980 µl of
eluent F, was subjected to anion-exchange chromatography on a MiniQ PC 3.2/3 (Amersham Pharmacia Biotech; eluent F: 10 mM
K2HPO4, pH 7.0; eluent G: 20 mM
K2HPO4, pH 7.0 with 1 M NaCl;
gradient: 0-5 min: 0% G, 5-35 min: 0-40% G, 35-37 min: 40-100%
G; flow rate: 30 µl/min).
Cell Proliferation Assay with Aortic Smooth Muscle Cells
Aortic smooth muscle cells (VSMCs) from normotensive
Wistar-Kyoto rats were subcultured in 96-well dishes (Falcon) at a
density of 5 × 104 cells/ml and kept in culture
medium containing 10% fetal calf serum (FCS) to reach a subconfluent
monolayer. After 24 h, the cells were growth-arrested in 0.1% FCS
for 48 h without affecting cell adherence to culture wells.
Quiescent VSMCs were then exposed to fresh culture medium with 0.1%
FCS with and without the tested agonists for another 48-h incubation
period. Cell proliferation was measured using the
[3H]thymidine incorporation rate as described elsewhere
(20). The viability of VSMCs was tested using trypan blue exclusion test. The viability was 95.2 ± 3.5% under control conditions and 93 ± 4.1% after stimulation with dinucleoside diphosphates.
Cell Proliferation Assay with Fibroblasts
Human skin fibroblasts were obtained from the Human Genetic
Mutant Cell Repository Institute for Medical Research (Camden, NJ) and cultured over several passages after detachment of the confluent cells with Puck's Saline A physiological solution (21) containing 0.04% trypsin and 0.02% EDTA buffer. The cells were allowed to grow as described for VSMCs.
Fibroblasts were seeded in 24-well culture plates and grown to
confluence. Then the medium was replaced by serum-free medium consisting of a mixture of Dulbecco's modified Eagle's medium and
Ham's F-10 medium (1:1). Following another 24-h cultivation in
serum-free medium, stimuli were added and cells were exposed to the
stimulating agents for 20 h before 3 µCi/ml
[3H]thymidine was added to the serum-free medium. Four
hours later, experiments were terminated by aspirating the medium and
subjecting the cultures to sequential washes with phosphate-buffered
saline containing 1 mM CaCl2, 1 mM
MgCl2, 10% trichloroacetic acid, and ethanol/ether (2:1,
v/v). Acid-insoluble [3H]thymidine was extracted into
250-µl dishes with 0.5 M NaOH, and 100 µl of this
solution was mixed with 5 ml of scintillant (Packard, Ultimagold) and
quantified using a liquid scintillation counter (Beckman LS 3801, Düsseldorf, Germany).
Human platelets were isolated (step 1) and deproteinated with
perchloric acid (step 2), and the supernatant nucleotides were concentrated by ion-pair reversed phase chromatography (step 3). In the
following steps, isolation and identification of dinucleoside diphosphates from human platelets is exemplified for
Gp2G.
After mononucleotides were separated from dinucleotides by affinity
chromatography (13) (step 4) the desalted and lyophilized eluate (step
5) was fractionated by anion-exchange chromatography (step 6). The
anion-exchange chromatogram is shown in Fig.
1A. Although
P1,P2-dinucleoside diphosphates have the same
charge, P1,P2-dinucleoside diphosphates
Ap2A, Ap2G, and Gp2G were separated because of hydrophobic interaction between the anion-exchanger and the
P1,P2-dinucleoside diphosphate.
6.07 ± 0.14 for Ap2A,
6.27 ± 0.25 for
Ap2G, and
6.91 ± 0.44 for Gp2G. At
least 61.5 ± 4.3% of the dinucleoside polyphosphates are
released by platelet activation. The intraplatelet concentrations
suggest that, in the close environment of a platelet thrombus, similar
dinucleoside polyphosphate concentrations can be found as in platelets.
Intraplatelet concentration can be estimated in the range of 1/20 to
1/100 of the concentration of ATP. In conclusion, Ap2A,
Ap2G, and Gp2G derived from releasable granules
of human platelets may play a regulatory role in vascular smooth muscle
growth as growth-promoting mediators.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
30 °C and
rethawed in bidistilled water (step 1). Then the resulting suspension
was deproteinized (step 2) with 0.6 M perchloric acid
(final concentration). After adjusting the pH to 7.0 with 5 M KOH, the precipitated protein and KClO4 were
removed by centrifugation.
80 °C (step 3).
80 °C.
30 °C. Fractions
corresponding to the main UV254 nm-absorbing peaks were
rechromatographed (step 8) on the reversed phase column (conditions as
in step 7).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, chromatogram of anion-exchange
chromatography of a platelet extract (column: TSK DEAE 5 PW, 20 cm,
150 × 20 mm, 10 µm, Tosohaas, Japan; eluent F: 20 mM K2HPO4 in water; eluent G: 20 mM K2HPO4 and 1 M NaCl
(pH 8.0) in water; gradient: 0-10 min, 0-5% G; 10-105 min, 5-35%
G; 105-110 min, 35-100% G; 110-120 min, 100% G; flow rate, 2.0 ml/min; fraction size, 2 ml; abscissa, retention time (min);
ordinate, UV absorption at 254 nm (arbitrary units)).
B, chromatogram of reversed phase chromatography of the
fraction labeled in A (column: Supersphere 100 C18
end-capped, 250 × 4 mm, particle size, 4 µm; flow rate, 0.5 ml/min; eluent A, 40 mM triethylammonium acetate in water;
eluent H, 100% acetonitrile; gradient: 0-4 min, 0-2% H; 4-55 min,
2-7% H; 55-60 min, 100% H; abscissa, retention time
(min); ordinate, UV absorption at 254 nm (arbitrary units)).
C, chromatogram of rechromatography of the fraction labeled
in B (conditions as described in legend of
B.).
Fractions of the anion-exchange chromatography with a significant absorbance at 254 nm were separated by reversed phase chromatography (step 7). In Fig. 1B the chromatogram of the reversed phase chromatography is given. The substance eluting at a retention time of 27 min was rechromatographed by reversed phase chromatography (step 8) using the same conditions as before (step 7).
In the last chromatographic step (step 8), a single UV peak was
obtained (Fig. 1C). The substance underlying this peak was identified by the following results: (a) The substance
chromatographed to homogeneity was analyzed by MALDI-PSD mass
spectrometry revealing a molecular mass of 709.4 (Fig.
2A). Each signal was assigned to a fragment of Gp2G as shown in Table
I. The MALDI-PSD spectrum was
completely identical to that of authentic Gp2G (14).
(b) The UV spectrum of guanine was obtained from the
rechromatographed substance, including the characteristic shift
obtained by acidification to pH 3.0, 7.0, and 9.0 (Fig. 2B;
Table II) (22). (c) The
retention time of the isolated fraction in step 8 was identical to that of authentic Gp2G (18). (d) Cleavage of the
molecules with 5'-nucleotide hydrolase (from C. durissus)
yielded GMP, as evidenced by MALDI mass spectra and by retention times
identical with those of authentic Gp2G. The cleavage
pattern was identical to that of synthetic Gp2G. Incubation
of the molecule with 3'-nucleotide hydrolase (calf spleen) and alkaline
phosphatase yielded no cleavage products. The enzymatic cleavage
experiments demonstrate that the polyphosphate chain interconnects the
guanosines via phosphoester bonds with the 5'-oxygens of the riboses
(Fig. 2C).
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In analogous manner also Ap2A as well as Ap2G were purified from human platelets and identified by the signal pattern of the PSD-MALDI-MS fragmentations (Table I), enzymatic cleavage experiments, and UV spectroscopy (Table II).
Ap2A, Ap2G, and Gp2G induced a
dose-dependent increase in DNA synthesis in vascular smooth
muscle cells as determined by [3H]thymidine uptake (Fig.
3). The bar labeled as
control in Fig. 3 represents the [3H]thymidine
incorporation in cultures without the stimulants. The maximum effect of
Ap2A was obtained at a concentration of 105
M, which induced an increase of vascular smooth muscle cell
proliferation of 225.9 ± 66.9% above control, 168.6 ± 31.0% above control at a concentration of 10
6
M for Ap2G, 77.0 ± 13.3% above control
at a concentration of 10
6 M for
Gp2G, and 1175.0 ± 66.3% above control at a
concentration of 5 × 10
9 M for PDGF.
These data are means ± S.E. from 10 independent experiments with
8 cultures. The raw data of a characteristic series of measurements for
each dinucleoside diphosphate were: 10
5 M
Ap2A: 16,440 ± 1,621 (control: 5,175 ± 160);
10
6 M Ap2G: 8,453 ± 2,210 (control: 3,199 ± 335); 10
6 M
Gp2G: 6,556 ± 429 (control: 3,986 ± 239) (in
cpm/well ± S.E.). Characteristic data for 10
8
M PDGF were 19,570 ± 2,193 (control 1,823 ± 219) (in cpm/well ± S.E.).
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The calculated EC50 (log M; mean ± S.E.)
for the P1,P2-dinucleoside diphosphates were
6.07 ± 0.14 for Ap2A,
6.27 ± 0.25 for
Ap2G,
6.91 ± 0.44 for Gp2G, and
9.72 ± 0.25 for PDGF. Costimulation with PDGF had no
significant effect on the threshold concentration of the
growth-stimulating effect of all three dinucleoside diphosphates. The
growth-stimulating effect of dinucleoside diphosphates on VSMCs was not
significantly modified in the presence of the platelet-derived growth
factor (PDGF).
In the range of 109 to 10
5 M
dinucleoside diphosphates did not significantly affect DNA synthesis of
cultured fibroblasts (10
5 M Ap2A:
178 ± 21; 10
5 M Ap2G:
221 ± 25; 10
5 M Gp2G:
229 ± 22; control: 172 ± 12 (in cpm/well ± S.E.)).
After isolation and identification of
P1,P2-dinucleoside diphosphates from human
platelets, the question arose as to whether P1,P2-dinucleoside diphosphates are released in
the extracellular space. Fig. 4 shows the
anion-exchange chromatograms of a platelet suspension (Fig.
4A) and a supernatant from an equivalent platelet suspension
aggregated with thrombin (Fig. 4B).
P1,P2-dinucleoside diphosphates can be found in
the supernatant after platelet aggregation (labeled in Fig.
4B by arrows) but not in the supernatant of
unstimulated platelets. The intracellular amount of
P1,P2-dinucleoside diphosphates in intact human
platelets can be estimated in the range of 0.5-2.0 attomol/platelet. From the concentrations determined in the
supernatant, the portion released upon platelet aggregation was
estimated as 61.5 ± 4.3% for each
P1,P2-dinucleoside diphosphates. The
intracellular amount of serotonin was 3.2 ± 0.5 attomol/platelet.
In the supernatant of unstimulated platelets serotonin was not
detectable. After platelet stimulation with thrombin the serotonin
amount of supernatant was 2.2 ± 0.4 attomol/platelet, indicating
that 68.7 ± 12.6% of the intracellular serotonin amount was
released by thrombin stimulation. The comparable degree of secretion of
P1,P2-dinucleoside diphosphates and serotonin
suggests that both classes of agents are released in a quantitatively
similar fashion.
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DISCUSSION |
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The findings revealed that P1,P2-dinucleoside diphosphates Ap2A, Ap2G, and Gp2G are endogenous messengers of human platelets.
The results of the cell proliferation assay show that Ap2A, Ap2G, and Gp2G act as potent growth mediators of VSMCs. The maximum effect of Ap2A, Ap2G, and Gp2G on VSMC proliferation rate was about one order of magnitude less, and the threshold concentration was about one order of magnitude higher than for PDGF, indicating that the dinucleoside phosphates are weaker growth factors than PDGF. Nevertheless, it has to be kept in mind that the local concentrations of these nucleotides after platelet aggregation probably are much higher than the physiological PDGF concentrations.
The receptor-mediating vascular growth is not yet known, although a P2 purinoceptor subtype is most likely. Especially, the P2Y2 purinoceptor may be considered, because ATP and GTP binding to this receptor cause similar mitogenic effects in VSMCs (23). At present, the growth-stimulating effect of dinucleoside diphosphates is only demonstrable in VSMCs. The growth of fibroblasts is not affected by dinucleoside diphosphates. This result may represent a different expression of purinoceptors on VSMCs and fibroblasts.
In contrast to ApnG and GpnG with n = 3-6 (7), Ap2A, Ap2G, and Gp2G do not potentiate the growth-stimulating effect of PDGF. Presently it is open to speculation whether this different behavior reflects activation of different purine receptor subtypes.
Because the P1,P2-dinucleoside diphosphates are released upon platelet activation, their growth-stimulating effect may contribute to that of PDGF and other growth mediators released from platelets. Therefore, together with known growth mediators, the described nucleotides may also participate in initiating atherosclerotic lesions.
Obviously, Ap2A, Ap2G, and Gp2G may exert their effects after release by platelet activation as is known for the diadenosine polyphosphates ApnA (with n = 3-6) (3, 24) and for the ApnGs and GpnGs with (n = 3-6) (7).
From the intracellular amount of P1,P2-dinucleoside diphosphates in intact human platelets, the intracellular concentration of P1,P2-dinucleoside diphosphates in intact human platelets can be calculated as 0.1-0.4 mM (volume of a platelet: 5.2 fl (25)). In platelets, two pools of nucleotides have been demonstrated (26). One pool is utilized for the metabolic needs of the platelets. The second pool, the dense granules, is a storage pool, which can be released into the extracellular space. As demonstrated, serotonin and dinucleoside diphosphates Ap2A, Ap2G, and Gp2G are released in parallel, it can be assumed that dinucleoside diphosphates are costored in dense granula with serotonin. The concentration of P1,P2-dinucleoside diphosphates in the dense granula can be estimated to be 0.2-0.8 mM, assuming that 50% of total volume of human platelets constitutes dense granula (27).
The extracellular dinucleoside polyphosphate concentrations occurring
after platelet activation depend on the extracellular volume of
distribution. The intraplatelet concentrations suggest that, in the
close environment of a platelet thrombus, similar dinucleoside
polyphosphate concentrations can be found as in platelets. Therefore,
the maximum extracellular concentration of
P1,P2-dinucleoside diphosphates can be
calculated as 0.2-0.8 mM in accordance to the
concentration of P1,P2-dinucleoside
diphosphates in dense granula. The minimum concentration can be
correspondingly estimated as 0.1 0.4 µM in
accordance with the concentration of
P1,P2-dinucleoside diphosphates after the
release into the surrounding blood volume of the platelets. Theses
estimations demonstrate that the extracellular concentrations of
P1,P2-dinucleoside diphosphates are sufficient for affecting the rate of proliferation of vascular smooth muscle cells.
How are these substances biosynthesized? The enzymes involved in
synthesis of diadenosine polyphosphates are only partially known, and
none of the known enzymes are described in human platelets (for review
see Ref. 28). Aminoacyl-tRNA synthetases catalyze the formation of
Ap3A and Ap4A (aminoacyl-AMP + ADP Ap3A, aminoacyl-AMP + ATP
Ap4A) (29).
Adenosine 5'-monophosphate does not react with this enzyme (30), and
therefore this type of enzymatic reaction cannot yield
Ap2A. Ap4A phosphorylases are another class of
diadenosine polyphosphate-synthesizing enzymes according to the
following reaction ADP + ATP
Ap4A + Pi
(29). Theoretically, the reaction of a diadenosine polyphosphate
phosphorylase catalyzing the formation of Ap2A should be
AMP + ADP
Ap2A + Pi. Alternatively, a
nonenzymatic synthesis may be considered. Given that mostly mononucleotides such as AMP are found together with biogenic amines such as catecholamines, the coexistence of both nucleotides and amines
within the same subcellular localization may allow a nonenzymatic reaction generating diadenosine polyphosphates. From AMP and a biogenic
amine a phosphoramidate may be generated, which is a highly reactive
intermediate. A further reaction with another AMP could then yield
diadenosine diphosphate (Ap2A). At present no definite
answer can be given by which biochemical pathway
P1,P2-dinucleoside diphosphates are synthesized
in human platelets.
In conclusion, releasable granules of human platelets contain
diadenosine diphosphate (Ap2A), adenosine guanosine
diphosphate (Ap2G), as well as diguanosine diphosphate
(Gp2G), which are potent growth-stimulating mediators in
vascular smooth muscle cells.
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ACKNOWLEDGEMENT |
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We thank A. Pacha for valuable technical assistance.
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FOOTNOTES |
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* This study was supported by a grant of the Deutsche Forschungsgemeinschaft (DFG: Schl 406/2-1).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Medizinische
Klinik IV: Nephrologie (WE 28), Universitätsklinikum Benjamin
Franklin, Freie Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany, Tel.: 49-30-8445-2441; Fax: 49-30-8445-4235;
E-mail: Hartmut.Schlueter@ruhr-uni-bochum.de.
Published, JBC Papers in Press, December 13, 2000, DOI 10.1074/jbc.M009527200
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ABBREVIATIONS |
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The abbreviations used are: VSMC, vascular smooth muscle cell; Ap2A, di(adenosine-5') diphosphate; Ap3A, di(adenosine-5') triphosphate; Ap4A, di(adenosine-5') tetraphosphate; Ap5A, di(adenosine-5') pentaphosphate; Ap6A, di(adenosine-5') hexaphosphate; Ap7A, di(adenosine-5') heptaphosphate; Ap2G, adenosine guanosine diphosphate; Gp2G, diguanosine diphosphate; TEAA, triethylammonium acetate; HPLC, high performance liquid chromatography; MALDI-MS, matrix-assisted laser desorption/ionization mass spectrometry; PSD, post-source decay; FCS, fetal calf serum.
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