Dinucleotides as Growth-promoting Extracellular Mediators

PRESENCE OF DINUCLEOSIDE DIPHOSPHATES Ap2A, Ap2G, AND Gp2G IN RELEASABLE GRANULES OF PLATELETS*

Joachim JankowskiDagger , Joost HagemannDagger , Martin TepelDagger , Markus van der GietDagger , Nina StephanDagger , Lars HenningDagger , Helena Gouni-Berthold§, Agapios Sachinidis, Walter ZidekDagger , and Hartmut SchlüterDagger ||

From the Dagger  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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 -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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 -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.

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 -80 °C (step 3).

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 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 -80 °C.

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 -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).

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).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


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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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Fig. 2.   Identification steps of the substance underlying the homogenous fraction labeled in Fig. 1C as Gp2G. A, positive-ion PSD-MALDI mass spectrum (abscissa: relative mass/charge, m/z, z = 1; ordinate: relative intensity). Interpretation of the spectrum is given in Table I. B, UV spectrum of the fraction (abscissa: wavelength/nm; ordinate: relative intensity, arbitrary units). C, chromatogram of anion-exchange chromatography of the homogenous fraction with 5'-nucleotidase (conditions: 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). Incubation with 3'-nucleotidase and alkaline phosphatase yielded no hydrolysis products.

                              
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Table I
Results of positive-ion PSD-Maldi mass spectra of the isolated fractions (A, adenine; A, adenosine; M, protonated parent ion; p, phosphate group, e.g. Ap4 adenosine tetraphosphate) (conditions: reflectron-type time-of-flight mass spectrometer (Reflex III, Bruker-Franzen, Germany); concentration of the analysed substance: 1-10 µmol/l; matrix solution: 50 mg/ml 3-hydroxy-picolinic acid; emission wavelength: 337 nm; pulse duration: 3 ns).

                              
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Table II
Maxima and minima values of the UV absorbance of the isolated fractions after two reversed phase chromatographies at different pH values (condition: UV/Vis-spectrophotometer DU-600 (Beckmann); pH values 3.0, 7.0, and 9.0 were adjusted by using 0.1 mol/l HCl and 0.1 mol/l NaOH respectively; scan speed: 400 nm/min).

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 10-5 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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Fig. 3.   [3H]Thymidine incorporation of vascular smooth muscle cells stimulated by various concentrations of diadenosine diphosphate (Ap2A), adenosine guanosine diphosphate (Ap2G), diguanosine diphosphate (Gp2G), and platelet-derived growth factor (PDGF). Abscissa, concentration in log M. The bars labeled as control represent the [3H]thymidine incorporation in cultures without the stimulants. Data are means ± S.E. from 10 independent experiments with 8 cultures.

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 10-9 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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Fig. 4.   Anion-exchange chromatography of a platelet suspension (A) and a supernatant from an equivalent platelet suspension activated by thrombin (B). The identity of P1,P2-dinucleoside diphosphates was determined by retention time comparison (conditions: column, Mono Q PC 3.2/2. 32 × 2 mm, Amersham Pharmacia Biotech (Sweden); eluent A: 20 mM K2HPO4, pH 8.0; eluent B: 20 mM K2HPO4 + 1 M NaCl, pH 8.0. Gradient: 0-100 min, 0-15% B; 100- 160 min, 15-40% B; 160-161 min, 40-100% B; 161-166 min, 100% B; flow rate, 0.5 ml/min) as well as matrix-assisted laser desorption/ionization mass spectrometry.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 right-arrow Ap3A, aminoacyl-AMP + ATP right-arrow 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 right-arrow Ap4A + Pi (29). Theoretically, the reaction of a diadenosine polyphosphate phosphorylase catalyzing the formation of Ap2A should be AMP + ADP right-arrow 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.

    ACKNOWLEDGEMENT

We thank A. Pacha for valuable technical assistance.

    FOOTNOTES

* 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

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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