J Biol Chem, Vol. 274, Issue 34, 23926-23931, August 20, 1999

Identification and Characterization of P¹,P⁷-Di(adenosine-5')-heptaphosphate from Human Platelets^*

Joachim Jankowski, Martin Tepel, Markus van der Giet, Ilva Maria Tente, Lars Henning, Ralf Junker, Walter Zidek, and Hartmut Schlüter^§

From the Medizinische Klinik I, Universitäts-Klinik Marienhospital, Ruhr University of Bochum, 44625 Herne, Germany and  Institut für Klinische Chemie und Laboratoriumsmedizin and Institut für Arterioskleroseforschung, Westfälische Wilhelms-University of Münster, 48149 Münster, Germany

	ABSTRACT

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Diadenosine pentaphosphate and diadenosine hexaphosphate have been isolated in human platelets and have been postulated to play an important role in the control of vascular tone. Here we describe the isolation and identification of diadenosine heptaphosphate from human platelets. Dinucleoside polyphosphates were concentrated by affinity chromatography from a nucleotide-containing fraction from deproteinated human platelets. Dinucleoside polyphosphates were purified by anion-exchange and reversed phase high performance liquid chromatography to homogeneity. Analysis of one of these fractions with matrix-assisted laser desorption/ionization mass spectrometry revealed a molecular mass of 1076.4 (1077.4 = [M + H]⁺) Da. UV spectroscopic analysis of this fraction showed the spectrum of an adenosine derivative. Comparison of the postsource decay matrix-assisted laser desorption/ionization mass spectrum of the fraction minus that of diadenosine heptaphosphate (Ap₇A) demonstrated that the isolated substance was identical to Ap₇A. The identity of the retention times of the authentic and the isolated compound confirmed this result. Enzymatic analysis demonstrated an interconnection of the phosphate groups with the adenosines in the 5'-positions of the riboses. With thrombin-induced platelet aggregation, Ap₇A is released from the platelets into the extracellular space. The vasoconstrictive action of Ap₇A on the vasculature of the isolated perfused rat kidney Ap₇A was slightly less than that of Ap₆A. The threshold of the vasoconstrictive action of Ap₇A was 10⁵ mol/liter. The vasoconstrictive effect was abolished by suramin and pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid, suggesting an activation of P_2x receptors. Furthermore, Ap₇A inhibits ADP-induced platelet aggregation. Thus, the potent vasoconstrictor Ap₇A derived from human platelets, like other diadenosine polyphosphates, may play a role in the regulation of vascular tone and hemostasis.

	INTRODUCTION

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Dinucleoside polyphosphates have attracted increasing interest with respect to their physiological role. Dinucleoside polyphosphates were identified in prokaryotic (1), eukaryotic, and mammalian cells (2).

Diadenosine tri- and tetraphosphate (Ap₃A and Ap₄A)¹ were the first dinucleoside polyphosphates to be identified in human platelets (3, 4). Ap₃A and Ap₄A were shown to act as vasodilators (5, 6). Diadenosine penta- and hexaphosphate (Ap₅A, Ap₆A) were identified and characterized as potent vasoconstrictors (7). Recently, further dinucleoside polyphosphates containing adenosine and guanosine (Ap_nG; n = 3-6) or containing two guanosines (Gp_nG; n = 3-6) were discovered in human platelets (8). Ap_nG (n = 5 or 6) are vasoactive also, whereas Gp_nG have no vasoconstrictive effect on the isolated perfused rat kidney. Both Ap_nG and Gp_nG are growth stimulators in vascular smooth muscle cells (8).

Recent studies suggested the existence of a further diadenosine polyphosphate (8). In this study, anion exchange chromatography revealed an unidentified UV absorbance at a retention time of 56 min.

In the present study, a diadenosine polyphosphate containing seven phosphate groups was isolated by testing chromatographic fractions of human platelet extracts for vasoconstrictive effects on the isolated perfused rat kidney. In the following, the identification of diadenosine heptaphosphate (Ap₇A) of human platelets and the characterization of the vasoactive effect of this substance is described.

	MATERIALS AND METHODS

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Chemicals-- HPLC water (gradient grade) and acetonitrile were purchased from Merck (Germany), and all other substances were from Sigma (Germany).

Purification of Dinucleoside Polyphosphates from Human Platelets-- First, human platelets were washed with an isotonic solution of NaCl and centrifuged (4000 rpm, 4 °C, 10 min) twice (step 1). The supernatant was aspirated, and the pellets were frozen to 30 °C. The platelet pellets were rethawed in bidistilled water (10 ml). The resulting suspension was deproteinated with 0.6 mol/liter (final concentration) perchloric acid and centrifuged (4000 rpm, 4 °C, 5 min) (step 2). After adjusting the pH to 7.0 with 5 mol/liter KOH, the precipitated proteins and KClO₄ were removed by centrifugation (4000 rpm, 4 °C, 5 min). 1 mol/liter triethylammonium acetate (TEAA) in water was added to the supernatant to a final concentration of 40 mmol/liter. The mixture was loaded to a preparative reversed phase column (step 3; Lichroprep RP-18 B, Merck; equilibration and sample buffer: 40 mmol/liter TEAA in water; flow rate: 5 ml/min). Nucleotides were eluted by 20% acetonitrile in water.

The lyophilized eluate of the reversed phase chromatography dissolved in 1 mol/liter ammonium acetate (pH 9.5) was loaded to a phenyl boronic acid resin (step 4). The resin was prepared according to Barnes et al. (9). The absorbed substances were eluted with 1 mmol/liter HCl (flow rate: 1 ml/min). The eluate from the phenyl boronic acid resin with 1 mol/liter TEAA to a final concentration of 40 mmol/liter added was desalted (step 5) by a reversed phase chromatography (Lichroprep RP-18 B, Merck; equilibration and sample buffer: 40 mmol/liter TEAA in water; flow rate: 5 ml/min). The lyophilized eluate was chromatographed (step 6) by anion exchange chromatography (TSK DEAE 5 PW 150 × 20 mm; Tosohaas (Japan); eluent A: 20 mmol/liter K₂HPO₄, pH 8; eluent B: 20 mmol/liter K₂HPO₄, pH 8, and 1 mol/liter NaCl in water; gradient: 0-10 min 0-5% B, 10-105 min 5-35% B, 105-110 min 35-100% B; flow rate: 2.0 ml/min). Fractions were collected according to UV absorbance at 254 nm (peak fractionation). Each fraction of the anion exchange chromatography with a significant UV absorbance at 254 nm was further chromatographed (step 7) on a reversed phase column (Superspher RP-18 end-capped, 250 × 4 mm, Merck; eluent A: 40 mmol/liter TEAA in water; eluent B: acetonitrile; gradient: 0-4 min 0-4% B, 4-64 min 4-11% B, 64-70 min 11-70% B; flow rate: 0.5 ml/min). Fractions with a significant UV absorption were rechromatographed using the conditions described (step 8).

Matrix-assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS)-- The lyophilized fractions from the reversed phase chromatography were examined by MALDI-MS and postsource decay (PSD)-MALDI-MS. A reflectron type time-of-flight mass spectrometer (Reflex III, Bruker, Germany) was used according to Hillenkamp and Karas (10). The sample was mounted on an x, y, z movable stage allowing irradiation of selected sample areas. In this study, a nitrogen laser (VSL-337 ND, Laser Science) with an emission wavelength of 337 nm and 3-ns pulse duration was used. The laser beam was focused to a typical diameter of 50 µm at an angle of 45° to the surface of a target. Microscopic sample observation was possible. 10-20 single spectra were accumulated for a better signal-to-noise ratio. In MALDI-MS, large fractions of the desorbed analyte ions undergo PSD during flight in the field free drift path. Using a reflectron type time-of-flight set-up, sequence information from PSD fragment ions of precursors produced by MALDI were obtained (11). Sample preparation for MALDI and PSD-MALDI experiments was identical. The concentrations of the analyzed substances were 1-10 µmol/liter in double distilled water. 1 µl of the analyte solution was mixed with 1 µl of matrix solution (50 mg/ml 3-hydroxypicolinic acid in water). To this mixture, cation exchange beads (AG 50 W-X12, 200-400-mesh, Bio-Rad) equilibrated with NH₄⁺ as counter ion were added to remove Na⁺ and K⁺ ions. The mixture was gently dried on an inert metal surface before introduction into the mass spectrometer. The mass accuracy was approximately 0.01%.

UV Spectrometry-- The substances purified to homogeneity by reversed phase chromatography were dissolved in 100 µl of water (pH 6.5) and were analyzed by a UV spectrometer (UV-visible spectrophotometer model DU-600, Beckman). The UV absorption was scanned from 400 to 190 nm with a scan speed of 200 nm/min.

Synthesis of Diadenosine Heptaphosphate-- Ap₇A was synthesized in a modification of the method of Ng and Orgel (12) using ATP and ATep as educts. A 10 mmol/liter concentration of each mononucleotide was mixed with 2.5 mol/liter 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in an aqueous solution containing 2 mol/liter HEPES and 50 mmol/liter MgCl₂. The pH was adjusted to 6.5. The reaction mixture was incubated for 24 h at 37 °C. The reaction products were concentrated on a preparative C18 reversed phase gel (Lichroprep RP-18 B, Merck). The nucleotides were eluted from the C18 gel by 30% acetonitrile in water. The eluate of the reversed phase chromatography was lyophilized and dissolved in 10 mmol/liter K₂HPO₄, pH 8 (eluent A) and was fractionated by an anion exchange column (Mono Q HR 10/10, Amersham Pharmacia Biotech (Sweden); eluent A: 20 mmol/liter K₂HPO₄, pH 8; eluent B: 20 mmol/liter K₂HPO₄, pH 8, with 1 mol/liter NaCl in water; gradient: 0-10 min 0-20% B, 10-145 min 10-40% B, 145-150 min 40-100% B; flow rate: 1.0 ml/min). Each fraction of the anion exchange chromatography showing a significant UV absorbance at 254 nm was desalted on a reversed phase column (Superspher RP-18 end-capped, 300 × 8 mm, Merck; eluent A: 40 mmol/liter TEAA in water; eluent B: 30% acetonitrile in water; flow rate: 1.0 ml/min). The fraction containing Ap₇A was identified by MALDI-MS.

Enzymatic Cleavage Experiments with Ap₇A-- Aliquots of the desalted and rechromatographed fractions of the anion exchange chromatography (step 8 of the purification procedure) were incubated with enzymes as follows. The samples were dissolved (a) in 20 µl of 200 mmol/liter Tris buffer (pH 8.9) and incubated with 5'-nucleotide hydrolase (3 milliunits; from Crotalus durissus, EC 3.1.15.1, from Roche Molecular Biochemicals, Germany; purified according to Sulkowski and Laskowski (13) 9 min at 37 °C); (b) in 20 µl of 200 mmol/liter Tris and 20 mmol/liter EDTA buffer (pH 7.4) and incubated with 3'-nucleotide hydrolase (1 milliunit; from calf spleen, EC 3.1.16.1, from Roche Molecular Biochemicals) 1 h at 37 °C; and (c) in 20 µl of 10 mmol/liter Tris, 1 mmol/liter ZnCl₂, and 1 mmol/liter MgCl₂ buffer (pH 8) and incubated with alkaline phosphatase (1 milliunit; EC 3.1.3.1, from calf intestinal mucosa, from Roche Molecular Biochemicals) 1 h at 37 °C. The reaction was terminated by an ultrafiltration with a centrifuge filter (exclusion limit of 10 kDa). After filtration of the enzymatic cleavage products, the filtrate, dissolved in 80 µl of eluent A, was subjected to the anion exchange chromatography (Mini Q PC 3.2/3; Amersham Pharmacia Biotech; eluent A: 20 mmol/liter K₂HPO₄, pH 8; eluent B: 20 mmol/liter K₂HPO₄, pH 8, with 1 mol/liter NaCl; gradient: 0-2 min 0-0% B, 2-27 min 0-40% B, 27-28 min 40-100% B, 28-30 min 100% B; flow rate: 0.1 ml/min.

Measurements of Perfusion Pressure in the Isolated Perfused Rat Kidney-- The effect of aliquots of the desalted and rechromatographed fractions of the anion exchange chromatography as well as authentic dinucleoside polyphosphates on vascular tone was evaluated in an isolated rat kidney perfused with a constant flow of 8-9 ml/min while perfusion pressure was continuously monitored. Details of the preparation are given elsewhere (14). The kidney was excised and immediately mounted into the perfusion system. The perfusion procedure generally followed the description given by van der Giet et al. (14). Briefly, the isolated rat kidney was perfused by a peristaltic pump in a single pass system with a solution containing 115 mmol/liter NaCl, 4.6 mmol/liter KCl, 1 mmol/liter CaCl₂, 1.2 mmol/liter MgSO₄, 1.2 mmol/liter NaH₂PO₄, 22 mmol/liter NaHCO₃, 49 mmol/liter glucose, and 35 g of gelatine/liter (Hemaccel; Behringwerke, Marburg, Germany) and equilibrated with 95% O₂, 5% CO₂. The perfusion medium and the kidney were kept constant at 37 °C. Perfusion flow was constant at 8-9 ml/min. Perfusion pressure was continuously monitored by a transducer (Gould P23) connected to a bridge amplifier (Hugo Sachs, Freiburg, Germany). Vasoconstrictor responses of the isolated perfused rat kidney to Ap₆A, Ap₇A, and ,-methylene ATP were assessed at basal tone after an equilibration period of 30 min. For each substance, dose-response curves were constructed, with 5 min allowed to elapse between consecutive doses. The nonspecific P₂ purinoceptor antagonist suramin (100 µmol/liter) and the P_2x purinoceptor antagonist pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid (PPADS, 30 µmol/liter) were added to the perfusate 30 min before challenge with mono- or dinucleotides. Furthermore, P_2x receptors were specifically occupied by continuous perfusion of the kidney with 10⁵ mol/liter ,-methylene ATP. This agent, which is a specific P_2x receptor agonist, is known to occupy P_2x receptor for any further activation.

Platelet Activation by Thrombin and Purification of Ap₇A from the Supernatant-- Three platelet concentrates were suspended in 300 ml of a 0.9% NaCl solution and divided into two parts. One of the parts was incubated with thrombin (0.05 units/ml) for 1 min. After this, the platelets were removed by centrifugation (4000 rpm, 4 °C, 10 min). The supernatant was deproteinated with 0.6 mol/liter (final concentration) perchloric acid and centrifuged (4000 rpm, 4 °C, 5 min). After adjusting pH to 7.0 with 5 mol/liter KOH, the precipitated proteins and KClO₄ were removed by centrifugation (4000 rpm, 4 °C, 5 min). The supernatants of both parts of the platelets concentrates were chromatographed according to steps 3-6. The dimension and gradient of the anion exchange chromatography were modified. Conditions were as follows: column: Mono Q PC 3.2/2, 32 × 2 mm, Amersham Pharmacia Biotech; eluent A: 20 mmol/liter K₂HPO₄, pH 8; eluent B: 20 mmol/liter K₂HPO₄ plus 1 mol/liter NaCl, pH 8; gradient: 0-2 min 0-0% B, 2-27 min 0-40% B, 27-28 min 40-100% B, 28-30 min 100% B; flow rate: 0.1 ml/min. Ap₇A was identified by retention time comparison with authentic Ap₇A as well as MALDI-MS. The concentration of Ap₇A was estimated using a calibration curve of Ap₇A.

Aggregometry-- Venous blood was obtained from healthy volunteers who had not used antiplatelet aggregation drugs for at least 2 weeks and was collected into heparinized sample tubes (Sarstedt, Germany). Platelet-rich plasma was separated from the blood sample by centrifugation at 1000 rpm for 10 min at room temperature. Platelets in the supernatant were counted on a Coulter MD II hematology analyzer (Coulter, Germany) and adjusted with platelet-poor plasma to 250,000 platelets/µl. Platelet-poor plasma was prepared from the pellet obtained in the first centrifugation step, after a further centrifugation at 4000 rpm for 10 min (room temperature).

Platelet aggregation studies were performed using the turbidimetric method according to Born (15) in a four-channel aggregometer (Labor, Germany). The instrument was adjusted with platelet-poor plasma to 100% and platelet-rich plasma to 0% of relative light transmission. Samples and reagents were prewarmed to 37 °C. Aggregatory effects of Ap₇A (final concentrations from 0.15 to 10 µmol/liter) were investigated using 10 µmol/liter ADP and 0.9% NaCl (DiaMed, Switzerland) as controls. Immediately after the addition of Ap₇A, ADP, or NaCl to platelet-rich plasma, relative light transmission was recorded.

For inhibition experiments, platelet-rich plasma was preincubated for 2 min with different amounts of Ap₅A, Ap₆A, and Ap₇A (up to 10 µmol/liter). After the addition of ADP (final concentration 1 µmol/liter), relative light transmission was recorded. Means ± S.E. of maximum aggregation (maximal relative light transmission) were calculated of 4-7 independent experiments.

	RESULTS

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Human platelets deproteinated with perchloric acid were concentrated by ion pair reversed phase chromatography. The affinity chromatography of the eluate by phenyl-boronic acid resin offers the possibility of the separation of mononucleoside and dinucleoside polyphosphates (8, 9). The desalted and lyophilized eluate was fractionated by anion exchange chromatography. The anion exchange chromatogram is given in Fig. 1A.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Purification of Ap7A from human platelets. A, anion exchange chromatography of a platelet extract dissolved in 5 ml of 20 mmol/liter K2HPO4 buffer (pH 8) in H2O (column: Tosohaas, TSK DEAE 5 PW 150 × 20 mm, Japan; eluent A: 20 mmol/liter K2HPO4, pH 8; eluent B: 20 mmol/liter K2HPO4, pH 8, and 1 mol/liter NaCl in water; gradient: 0-10 min 0-5% B, 10-105 min 5-35% B, 105-110 min 35-100% B; flow rate: 2.0 ml/min). B, reversed phase chromatography of the fraction labeled in A on an analytical reversed phase high performance liquid chromatographic column (Superspher RP-18 end-capped, 250 × 4 mm, Merck; eluent A: 40 mmol/liter TEAA in water; eluent B: acetonitrile; gradient: 0-4 min 0-4% B, 4-64 min 4-11% B, 64-70 min 11-70% B; flow rate: 0.5 ml/min). C, rechromatography of the fraction labeled in B (conditions are the same as used in B).

The molecular mass of underlying substances of each peak was determined by MALDI-MS. All except the peak labeled by an arrow in Fig. 1A belong to known dinucleoside polyphosphates like Ap₃A (3), Ap₄A (4), Ap₅A, and Ap₆A (7) as well as Ap_nG and Gp_nG (with n = 3-6) (8). Only the labeled peak (Fig. 1A) had not been identified earlier and had a vasoconstrictive effect in the isolated perfused rat kidney. Therefore, in the following only the identification of the peak at 86.8 min (Fig. 1A) is described.

Fig. 1B shows a typical chromatogram of a reversed phase chromatography from the peak labeled in Fig. 1A. The substance eluting at a retention time of 56 min had a vasoconstrictive effect on the isolated perfused rat kidney (data not shown). Therefore, this fraction was rechromatographed by reversed phase chromatography using the same conditions as before. In the last chromatographic step, a single UV peak was obtained (Fig. 1C).

The UV spectrum of the isolated fraction was essentially identical to that of adenosine, confirming that a nucleotide containing adenosine was present in the isolated fraction (data not shown).

In Fig. 2A, the PSD-MALDI-MS spectrum of the fraction labeled by an arrow in Fig. 1C is shown. The molecular mass of the isolated substance was determined as 1076.4 (1077.4 = [M + H]⁺) Da by this PSD-MALDI-MS spectrum (Fig. 2A). This molecular mass as well as the fragments corresponds to that of authentic Ap₇A (Fig. 2B). Retention time of the isolated fraction (Fig. 3A) was identical to that of authentic Ap₇A (labeled in Fig. 3E). To answer the question how the phosphoesters are connected to the riboses of the adenosines, the products generated by nucleotide hydrolase digestion and alkaline phosphatase were analyzed. Incubation of the isolated molecules with alkaline phosphatase (Fig. 3B) or 3'-nucleotide hydrolase (calf spleen) (Fig. 3C) yielded no cleavage products. When Ap₇A recovered from biological extracts was treated with 5'-nucleotide hydrolase (Crotalus durissus), the initial peak of Ap₇A decreased, and the hydrolysis products AMP and adenosine hexaphosphate appeared (Fig. 3D). The cleavage pattern was identical with that of authentic Ap₇A (Fig. 3E). The results of the enzymatic cleavage experiments demonstrate that the polyphosphate chain interconnects the two adenosines via phosphoester bonds to the 5'-positions of the riboses.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Positive-ion PSD-MALDI mass spectrum of the fraction labeled in Fig. 1C by an arrow (A) and authentic Ap7A (B). A', adenine; A, adenosine; M, protonated parent ion; p, phosphate group (e.g. Ap4, adenosine tetraphosphate). Abscissa, relative mass/charge (m/z, z = 1); ordinate, relative intensity in arbitrary units.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Enzymatic cleavage experiments. Anion exchange chromatograms of the fraction of reversed phase rechromatography labeled in Fig. 1C before (A) and after incubation with alkaline phosphatase (B), with 3'-nucleotidase (C) and with 5'-nucleotidase (D). E shows the chromatogram of authentic Ap7A after incubation with 5'-nucleotidase. Conditions were as follows: column: Mono Q PC 3.2/2, 32 × 2 mm, Amersham Pharmacia Biotech; eluent A: 20 mmol/liter K2HPO4, pH 8; eluent B: 20 mmol/liter K2HPO4 plus 1 mol/liter NaCl, pH 8; gradient: 0-2 min 0-0% B, 2-27 min 0-40% B, 27-28 min 40-100% B, 28-30 min 100% B; flow rate: 0.1 ml/min.

After platelet aggregation with thrombin, Ap₇A is released into the extracellular space. Fig. 4 shows the anion exchange chromatogram before (Fig. 4A) and after platelet aggregation with thrombin (Fig. 4B). Ap₇A can be found in the supernatant after platelet aggregation (labeled in Fig. 4B by an arrow). The extracellular concentration of Ap₇A can be estimated by the UV peak in the range of 0.05-0.1 µmol/liter after thrombin stimulation. The concentration of Ap₇A in the supernatant of untreated platelet extract was lower than the detection limit (detection limit was <5 nmol/liter; Fig. 4).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Anion exchange chromatography of a platelet suspension without (A) and with (B) aggregation with thrombin. The identity of Ap7A was determined by retention time comparison (conditions as follows: column: Mono Q PC 3.2/2, 32 × 2 mm, Amersham Pharmacia Biotech; eluent A: 20 mmol/liter K2HPO4, pH 8; eluent B: 20 mmol/liter K2HPO4 plus 1 mol/liter NaCl, pH 8; gradient: 0-2 min 0-0% B, 2-27 min 0-40% B, 27-28 min 40-100% B, 28-30 min 100% B; flow rate: 0.1 ml/min) as well as matrix-assisted laser desorption/ionization mass spectrometry.

Furthermore, the amounts of diadenosine polyphosphates released were determined in relation to the total amounts. The portion released upon platelet aggregation was estimated as 60% for each diadenosine polyphosphate.

At basal tone, Ap₇A caused a dose-dependent vasoconstriction (Fig. 5A). The vasoconstrictor EC₅₀ value (mol/liter) of Ap₇A ((5.34 ± 1.77) × 10⁴) was higher than the EC₅₀ value of Ap₅A ((1.07 ± 0.05) × 10⁵), Ap₆A ((1.86 ± 0.28) × 10⁵), and ,-methylene ATP ((8.08 ± 0.53) × 10⁶) (Fig. 5A). The minimal effective concentration of Ap₇A was less than 10⁵ mol/liter. Ap₇A is less effective on the vascular resistance of the isolated perfused rat kidney than ,-methylene ATP, Ap₅A, or Ap₆A.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   A, change in perfusion pressure in the isolated perfused rat kidney induced by Ap7A (), Ap6A (), Ap5A (), and ,-methylene ATP (). B, change in perfusion pressure in the rat isolated perfused rat kidney induced by Ap7A in the absence () and presence () of PPADS (30 µmol/liter). C, change in perfusion pressure in the rat isolated perfused rat kidney induced by Ap7A in the absence () and presence () of suramin (100 µmol/liter). Abscissa, concentration of agonist (log mol); ordinate, change in perfusion pressure (mm Hg)). Each point is the mean of at least six determinations (n = 6), and vertical lines show the means ± S.E.

In the presence of suramin (100 µmol/liter) and PPADS (30 µmol/liter), the responses of kidney vasculature to Ap₆A and Ap₇A were completely abolished. The response of Ap₇A in the presence and absence of PPADS and suramin is shown in Fig. 5, B and C, respectively. Incubation of the isolated perfused rat kidney with ,-methylene ATP abolished the vasoconstrictive effect of Ap₇A (Fig. 6). Since no vasodilator response to Ap₇A was seen, even after vascular tone was increased by ,-methylene ATP, no further antagonists specific for P2Y receptors were tested. Conversely, the vasoconstrictive effect of angiotensin II was not affected under continuous perfusion with ,-methylene ATP.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Changes in perfusion pressure of the isolated perfused rat kidney induced by bolus injections of Ap7A (103 mol/liter), ,-methylene ATP (106 mol/liter), or angiotensin (107 mol/liter) before (open column) and after (solid column) P2x receptors of the rat perfused kidney were specifically occupied by continuous perfusion with 105 mol/liter ,-methylene ATP. Each column represents the mean of at least six determinations (n = 6), and vertical lines show means ± S.E.

Ap₇A has no direct effect on aggregation of isolated platelets (data not shown). However, the ADP-induced platelet aggregation is inhibited by Ap₇A. The concentration dependence of Ap₇A on the inhibition of ADP-induced platelet aggregation is shown in Fig. 7. The IC₅₀ value of Ap₇A on the inhibitory effect of ADP aggregation was calculated at (2.94 ± 0.88) × 10⁶ mol/liter; the value for Ap₆A was (2.68 ± 0.74) × 10⁶ mol/liter; and the value for Ap₅A was (4.06 ± 0.92) × 10⁶ mol/liter.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Inhibition of ADP-induced platelet aggregation by Ap5A (), Ap6A (), and Ap7A (). ADP (final concentration 1 µmol/liter) was added to platelet-rich plasma after a 2-min preincubation with different amounts of Ap5A, Ap6A, or Ap7A. As control (), 0.9% NaCl was used. Results are given as means ± S.E. of maximum aggregation (maximal relative light transmission) of four independent experiments (n = 4).

	DISCUSSION

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In the last decade, diadenosine polyphosphates Ap_nA (n = 3-6) received considerable attention in view of their multiple biological and pharmacological activities. Besides their role in platelet aggregation (16), diadenosine polyphosphates have potent vasoconstrictive and vasodilator properties. The vasoactive actions of diadenosine polyphosphates were demonstrated in numerous vascular models (17).

Comparison of the various diadenosine polyphosphates reveals that the action of these compounds on vasculature depends on the number of phosphate groups. Accordingly, vasodilation can be observed in intact vessels after administration of Ap₂A, Ap₃A, and Ap₄A, whereas Ap₅A and Ap₆A are vasoconstrictive (17). Our results demonstrate for the first time a diadenosine polyphosphate with a vasoconstrictive effect with seven phosphate groups. The vasoconstrictive effect of Ap₇A is comparable with the effect of Ap₅A, the diadenosine polyphosphate with the most intensive vasoconstrictive effect known (7).

To the best of our knowledge, unlike diadenosine polyphosphates Ap_nA with n = 3-6, Ap₇A has not been isolated from any tissue so far. The question arises of how this substance is biosynthesized. In general, four classes of enzymes are known to synthesize dinucleoside polyphosphates in vitro (for a review, see Ref. 18): Ap₄A phosphorylases, luciferases, guanylyl transferases, and the aminoacyl-tRNA synthetases. However, these enzymes are not capable of producing of dinucleoside polyphosphates in vivo. In vivo Ap₄A is enzymatically synthesized from ATP and lysine by lysyl-sRNA synthetase. This pathway was shown using baby hamster kidney cells, salmonella, and Escherichia coli (18). Principally, this pathway is also capable of producing Ap₇A.

Alternatively, a nonenzymatic synthesis may be considered. Given that mostly mononucleotides such as ATP are found together with biogenic amines such as catecholamines, the coexistence of both nucleotides and amines within the same subcellular localization may be due to the type of nonenzymatic reaction generating diadenosine polyphosphates. From ATP and a biogenic amine, a phosphoramidate may be generated, which is a highly reactive intermediate. A further reaction with another ATP could then yield Ap₆A. However, also diadenosine polyphosphates with a higher length of the phosphate chain could be produced by this reaction, if an additional step to add phosphates to the phosphoramidate is assumed.

The experiment with the P₂ purinoceptor antagonist, suramin, and the P_2x purinoceptor antagonist, PPADS, suggests that the vasoconstrictive action of Ap₇A is due to activation of P_2x purinoceptors. During perfusion with maximally effective concentrations of ,-methylene ATP, Ap₇A did not further increase vascular resistance. These experiments further support the view that Ap₇A produces vasoconstriction via P_2x purinoceptors. Because P_2x1 receptors have been identified in rat kidney (19) and ,-methylene ATP is a specific agonist for the P_2x1 subtype (20), the vasoconstrictive effect of Ap₇A is probably mediated by the P_2x1 subtype.

Principally, Ap₇A appears to act by the same mechanisms as Ap₅A and Ap₆A (14). The latter diadenosine polyphosphates were shown to exert vasoconstriction via the P_2x purinoceptor, whereas Ap₂A and Ap₃A are vasodilators, acting via A₂ receptors (5). Chan et al. (19) further characterized the P_2x receptor in rat kidney vasculature as the P_2x subtype. Present results strongly suggest that Ap₇A also activates this P_2x purinoceptor subtype. Compared with Ap₅A and Ap₆A, its receptor affinity as estimated from the EC₅₀ value appears to be less, suggesting that the optimal length of the phosphate chain to activate the P_2x purinoceptor may be five phosphates.

Because of the different vascular effects of diadenosine polyphosphates, depending on the number of phosphate groups, it may be speculated that for both adenosine moieties in the molecules an optimal distance exists to have a vasoconstrictive effect. The EC₅₀ value of Ap₅A and Ap₆A indicates a slightly higher receptor affinity of Ap₅A and Ap₆A than of Ap₇A, suggesting that the optimal distance between the adenosine moieties of the known endogenous diadenosine polyphosphates is achieved with five phosphate groups.

After the biochemical characterization, the question arose of whether the concentration range of Ap₇A used in the isolated perfused rat kidney is physiologically relevant. Compared with ATP, which can easily be identified in chromatograms of platelet extracts, the concentration of Ap₇A may be in the range of to ATP. Earlier experiments showed that about 80% of diadenosine polyphosphates of platelets are released during aggregation (21). Obviously, the diadenosine polyphosphate concentration occurring in extracellular space after platelet aggregation depends on the volume of distribution, but the intraplatelet concentrations suggest that in the close environment of platelet thrombus similar diadenosine polyphosphate concentrations can be found as in platelets. In particular, platelets and chromaffin granules of the adrenal medulla have rather high concentrations of diadenosine polyphosphates (Ap_nA; n = 3-6), which was up to 6 mmol/liter. Local extracellular concentration of up to 100 µmol/liter have been assumed (22).

The physiological role of Ap₇A is still unclear. Like Ap_nA (n = 3-6), (3, 4, 8), Ap₇A is released by platelet aggregation.

Furthermore, Ap₇A may thereby reach extracellular concentrations sufficient to affect vasculature, as estimated from the concentration-response curves in the isolated perfused rat kidney. The experiments revealed a lower EC₅₀ of Ap₇A compared with Ap₅A and Ap₆A. Obviously, in the sigmoidal part of the concentration-response curve, Ap₇A has weaker effects than Ap₅A and Ap₆A. However, this does not necessarily imply that the extracellular effects of Ap₇A are negligible; e.g. due to the long phosphate chain, the affinity of hydrolases may be lower and hence the extracellular half-life may be longer than that of the other diadenosine polyphosphates. Therefore, Ap₇A may be involved in local changes of perfusion induced by platelet aggregation.

Ap₇A inhibits ADP-induced platelet activation which is mediated via the P2Y1 and P2T_AC receptor (23). Inhibition of platelet aggregation by diadenosine polyphosphates was proposed to be due to competition between the dinucleotides and ADP at a specific receptor site on the platelet membrane (24), which, according to experiments with a recombinant P2Y1 receptor, is not identical with the latter (25). Therefore the inhibitory action may be developed at the P2T_AC receptor. Ap₅A has the highest inhibitory activity on platelet aggregation, followed by Ap₆A and Ap₇A. This is in good agreement with the results of Harrison et al. (24).

Like the other diadenosine polyphosphates, Ap₇A may not only occur in platelets, but also in adrenal medulla and sympathetic nerve endings, although no attempts have been made to isolate Ap₇A from cells other than platelets. Assuming the same widespread occurrence of Ap₇A as of the other analogues, a role as sympathetic co-transmitter may be discussed. However, the physiological concentrations of Ap₇A appear to be lower than those of Ap₅A and Ap₆A, at least in human platelets. At present, potential differences in the effects of Ap₅A, Ap₆A, and Ap₇A have not been studied yet.

In summary, Ap₇A isolated from human platelets appears to be a potent vasoconstrictor whose vasoconstrictive effect is mediated by a P_2x receptor, possibly by the P_2x1 subtype of this receptor. Furthermore Ap₇A inhibits platelet aggregation.

	ACKNOWLEDGEMENT

We thank B. Theiling for valuable technical assistance.

	FOOTNOTES

^* This study was supported by Deutsche Forschungsgemeinschaft Grants Schl 406/2-1 and 2-2.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: Univ.-Klinik Marienhospital der Ruhr-Universität Bochum, Hölkeskampring 40, 44625 Herne, Germany. Tel.: 49-2323-499-1696; Fax: 49-2323-499-302; E-mail: Hartmut.Schlueter@ruhr-uni-bochum.de.

	ABBREVIATIONS

The abbreviations used are: Ap₃A, P¹,P³-di(adenosine-5')-trisphosphate; Ap₄A, P¹,P⁴-di(adenosine-5')-tetraphosphate; Ap₅A, P¹,P⁵-di(adenosine-5')-pentaphosphate; Ap₆A, P¹,P⁶-di(adenosine-5')-hexaphosphate; Ap₇A, P¹,P⁷-di(adenosine-5')-heptaphosphate; PPADS, pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; ATep, adenosine-5'-tetraphosphate; TEAA, triethylammonium acetate; PSD, postsource decay; HPLC, high performance liquid chromatography.

	REFERENCES

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