MECHANISM OF THE PERSISTING TxA2 RECEPTOR ANTAGONISM BY PICOTAMIDE

ThrombosisResearch,Vol.85,No.3,pp.207-215,1997 Copyright@ 1997Elsevie.rScienceLtd Printedin theUSA. Allrightsreserved 0049-3848/97 $17.GO + .00

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MECHANISM OF THE PERSISTING TxA2 RECEPTOR ANTAGONISM BY PICOTAMIDE Fabio M. Pulcinelli, PasqualePignatelli,Marzia Pesciotti,Silvia Sebastiani, * Simona Parisi and Pier Paolo Gazzaniga. Dept. of ExperimentalMedicineand Pathology,Universityof Rome La Sapienza,Viale Regina Elena 324, 00161 Roma, Italy; *LPB ResearchInstitute,Via dei Lavoratori54, CiniselloBalsamo(Ml), Italy. (Received28August 1996 by EditorG.E Gensini;revised/accepted22November1996)

Abstract Picotamideis a dual TxA2 receptorantagonist/TxA2 synthetaseinhibitor. As the picotamide effect is maximumonly after 5-10 min of incubation, aim of the present work was to study whether the effect of picotamide could be observed even after wash out of the drug from the external buffer. Platelet aggregation, serotonin release and changes in intracellular calcium were analyzed in platelets treated with picotamide and then gel-filtered, in comparison with gel-filtered platelets treated with picotamide. To exclude the possibility that the inhibitory effect of picotamide is due to its intracytosolicstorage, serotonin release in digitonin-permeabilizedplatelets was measured. Platelet aggregation and serotonin release in response either to U46619, a synthetic agonist of the thromboxane A2 receptor, or arachidonic acid or collagen,as well as the changes in intracellular calcium concentration after U46619 or arachidonic acid stimulation, were inhibited by picotamide even when it had been washed out by gel-filtration. Both inhibitions were very similar to that observed in experiments in which picotamidewas present in the medium.As the serotonin release in digitonin permeabilized platelets presented the same inhibition it may be excluded that picotamide effect is consequentupon the cytosolic storage of the drug. Since our results clearly indicatethat the action of picotamide persists even after washing out of the drug from the medium, the idea that this antagonistic effect may be dependent on its binding to platelet plasma membrane rather than on its cytosolic concentration, is strongly substantiated. Copyright 01997E.kevier Science L/d —————

~~y words: Antiplateletdrugs,TxA2 receptor,Picotamide,PlateletActivation. Corresponding author: Prof. Pier Paolo Gazzaniga, Dept. of Experimental Medicine and Pathology,UniversityLa Sapienza,Viale Regina Elena 324, 00161 Roma, Italy. 207

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Picotamide (G137 or N,N’-bis-3-picoly l-4-methoxy-isophthalamide) is a dual thromboxane Az (TxAz)-synthetase inhibitor/thromboxane endoperoxide receptor antagonist, that inhibits in vitro platelet aggregation,degranulation,and thromboxane 62 production, in a dose dependent manner, in response to several agonists acting via TxA2 formation, such as collagen, arachidonicacid (AA), the synthetic agonist of TxA#cyclic endoperoxidereceptor U46619and ADP (1, 2). .Exvivo studies showed a reduction of platelet aggregation and TxA2 production after ADP, collagen and PAF stimulation in subjectstreated with picotamide(1200 mg/die)(3). 3H-pico@m& binds to platelet TxA2 receptors with a Kd of 325 nM; TxA2 WM109S U46619 and ONO11120 are able to displacethe picotamidebinding;contrary to wh.al happens with TxB2, prostacyclin and prostagJandinE2,the displacement effect Is reduced if the incubation time with picotamideis prolongedfor more than 30 min (4). Moreover, picotamide can directly inhibit the in vitro binding of [SH]-U46619 to the platelet membrane (5), and the ex vivo binding of [1251]- PTA - OH (9,11- dimethylmethano-11,12-methane-16-(3-4-hydroxyphenyl)-l 3, 14-dihydro-13-aza-15-tetranorTxA2) 2 and 4 hours after a single administrationof 300 mg of picotamide(6). In a previous study (7) we demonstratedthat picotamidein gel-filteredplatelets and in thrombin degranulated platelets (10-4 M) significantly reduced the intracellular calcium concentration changes and phospholipaseC activation induced by U46619, AA and ADP. Picotamideeffect was evidentafter 5 min of preincubationas regardsthe calcium response (7). SimiJarly,the effect of picotamideon platelet aggregation needs 10 min of preincubation (2); moreover its binding seems to be reversible in a first phase and stable in a time dependent manner, reaching the maximum level after .20 min (4). Aim of the present work was to study whether the effect of picotamide could be observed even after its wash out from the external buffer. At this purpose we have analyzed aggregation, serotonin release and changes in intracellular calcium concentration induced by the synthetic analog of TxA2 U46619, or arachidonic acid (AA), in platelets pretreatedwith picotamide(10-4M for 10 min at 37°C) and then gelfiltered, in comparison with gel-filtered platelets (GFP) subsequently treated with picotamide,.To exclude the possibility that the inhibitory effect of picotamide was due to its intracytosolic storage, serotonin release was measured in digitoninpermeabilized platelets . The results support the idea that picotamide is capable to inhibit all the above mentioned effects even after washing out and that its effect is not dependent on intracytosolicstorage. MATERIALSAND METHODS P/ate/etpreparation. Platelet Rich Plasma (PRP) was obtained after centrifugation(180 x g for 15 rein) of blood samples taken by venipuncture from informed healthy volunteers using acid/citrate/dextrose(ACD) (8) as anticoagulant(1:7 v/v). PRP was then centrifuged (800 x g for 10 rein) to concentrate the platelets (6 x 108 cells/ml). Concentrated platelets were incubated with the fluorescent indicator of intracellular free calcium concentration Fura-2-AM (4 wM) (MolecularProbes Eugene, OR, USA) for 30 min at 37° C and 15 min at room temperature,or [14C]Serotonin (lmM) (ICN, Costa Mesa, CA, USA) for 1 h at 37”C. Picotamidetreated platelets were prepared by incubating platelets for 20 min with 10-4M picotamide(kindly providedby SAMIL, Sandoz Group, Rome, Italy) during Fura-2-AMor 14CSerotonin incubation.The external dye Fura-2AM, 14c serot~nin and picotamide were separated from platelets by gel-filtration (gel-

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filtered-platelets-GFP)on Sepharose26 (Pharmacia,Uppsala, Sweden) using Caz+free Tyro.de’sbuffer containing0.29’.albumin (Bovineserum albumin fraction V -BSASigma Chemical Co., St. Louis, MO, USA), O.IYOglucose and 10 mM HEPES (pH 7.35). Ali the described procedureswere completedwithin 3 hours from blood collection. Platelet Aggregation /n vitro platelet aggregation was evaluated according to Born (9), in a four sample

3210 Aggrecorder (Menarini Diagnostic, Florence, Italy), using siliconized glass cuvets, at 37°C and under continuous stirring at 1000 rpm. 500 nM U46619 (Sigma Chemical Co.), 2 p.g/ml collagen and 150 PM arachidonic acid (AA) (both from Menarini Diagnostic), were used as aggregation inducers. Platelets were resuspendedat the concentrationof 2.!5x 10scells/ml and fibrinogen (1 mg/ml, Sigma Chemical Co.) was added before the agonists. Serotonin Re/ease

Platelet secretion was evaluated by measuring the release of 14C-serotonin and expressedas percentageof total 14C-serotonincontent. Activation of 14C-serotonin Iabelled platelets was stopped after two min with formaldehyde/EDTA according to the method of Costa and Murphy (10); after centrifugation(5000 x g for 1 rein)the radioactivityof supernatantwas measured using a LKB (Pharmacia, Uppsala, Sweden) liquid scintillationcounter. Changes in intraplatelet calcium concentration.

AA (Menarini Diagnostic) or the synthetic agonist of endoperoxide-thromboxane receptor U46619 (Sigma Chemical Co.) were added to cuvets containing 0.5 ml of Fura 2-loaded GFP or picotamide pretreated platelets (2 x 108cells/ml). The cuvets were thermostatically regulated at 37°C and stirred continuously. Fluorescence changes were monitored with a Kontron (Zurich, Switzerland)SFM 25 fluorimeter, set at 340 nM excitation and 510 nM emission.Differencesin free calcium concentrations (A) between the unstimulatedand stimulatedGFP were calculated using as Fmin the value determined after the addition of digitonin (50 PM) (Sigma Chemical Co.) in the presence of EGTA (2 mM) and Tris base (20 mM), as Fmax the value measured after the addition of excess CaClz (10 mM), and a Kd of 224 nM after correction for extracellular dye (11). AO/O was then calculated as ratio between A and baseline values. Digitonin permeabilized platelets

To permeabilize platelets digitonin method was used, as digitonin produces in platelet plasma membranes holes larger than those producedby electropermeabilization(12). Platelets, preincubated with 14C Serotonin and picotamide, after washing by gelfiltration using permeabilizationbuffer (140 mM NaCl, 14 mM Mg C12,5 mM glucose, 1 mM EGTA, 0.37°A BSA, 10 m M Ieupeptine and 10 mM HEPES, pH 6.5), were permeabilized with digitonin (10 PM) at 30°C for 15 min. Cell suspensions were washed by centrifugation, and permeabilized platelets were resuspended in a new buffer (140 mM potassiumglutamate,17 mM MgC12,10 mM EDTA, 10 mM Ieupeptine, 5 mM ATP, 14 mM GTP, 10 mM HEPES, pH 7.35) at the concentration of 1X108 cells/ml. To assess the rate of platelet permeabilization as well as the functional integrity of the digitonin-permeabilizedplatelet preparationswe used the method first

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proposed by Haslam and Davidson (14), using varying concentrations of calcium; in fact, at lowest calcium concentrations,serotoninreleaseis minimal also in response to agonists, while at high calcium concentrationsplatelets release serotonin even in the absence of agonists, thus indicatingthe unpermeabilizedcell rate. Different series of digitonin-permeabilized platelets were equilibrated for 5 min at room temperature with varying calcium concentrations(pCa from -7.2 to -3); the free calcium concentrations were calculated by means of a computer program (13); then the different series were activated with U46619 or 0.5 U/ml thrombin; as control one series was treated with the agonistsolventfor 15 min at room temperature. Permeabilized platelet preparations, unresponding to thrombin or unable to reach 90Y0of permeabilization,were discarded. In each sample the release of IAC Iabelledserotoninwas measured after the addition of formaldehyde/EDTA(1O)to stop plateletactivation;after centrifugation(5000 x g for 1 rein) the radioactivityof supernatantwas measuredas above described. RESULTS P/ate/et aggregation

The results of platelet aggregation in response to 500 nM U46619, 150 PM Arachidonic Acid and 2 ~g/ml collagen, either in platelets pretreated with picotamide (10-4 M for 20 min at 37°C) and then gel-filtered or in gel-filtered platelets (GFP) incubatedwith picotamide20 min beforethe agonistaddition,are reported in Table 1. The results show a similar inhibition in both preparations after stimulation with U46619, AA or collagen. Both platelet preparations aggregated in responseto thrombin at the same extent of control gel-filtered platelets (data not shown). Serotonin release

Serotonin release induced by 500 nM U46619, 150 KM ArachidonicAcid and 2 pg/ml collagen in platelets pretreated with picotamide (10-4M for 20 min at 37°C) and then gel-filtered or in GFP incubatedwith picotamide20 min before the agonist addition, is reported in Fig. 1. The results show an evident inhibition of platelet degranulationin platelets pretreated with picotamide and then gel-filtered,as well as in gel-filtered platelets treated with Table1 Maximumpercentagesof plateleta gregationinducedbydifferentagonistseitherin plateletspretreatedwithpicotamide10-1 ? Mfor20minat37°C)andthengel-filteredor in gel-filtered platelets (GFP)incubatedwith picotamide20 min beforethe agonist addition. Agonist

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Dataare expressedas meanpercentagesi S.E.Miof 5 experimentsperformed. Datawereanalyzedby Studentt testforpaireddata. (*=p <0.03. ● *=p< 0.005.)

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Serotoninreleaseinducedby500nMU46619,150yM ArachidonicAcidand2 ~g/ml collagenin platelets pretreatedwith picotamide(10-4M for 20 min at 37”C) and then cl-filteredor in GFP incubatedwithpicotamide20 minbeforethe agonistaddition. ?he resultsare expressedas meanpercentage* S.E.M.of 4 experimentsperformed. Data were analyzedby Studentt test for paireddata. (*= P <0.05. **=p < (3.02.)

picotamide 20 min before the agonist addition. In fact, in the first preparation the release of 14C-5HT was 28.6A4.2°/0versus 58.6*11.6 !40of untreated platelets after stimulation with 500 nM U46619,32.Oti.5Y0 versus 64.3f12.4Y0after stimulation with 150 KM AA and 39.3t3,7°/0versus 67.3t10.1°/0,after stimulationwith 2 Ug/mlcollagen. The same inhibition of serotonin release was observed in gel-filtered platelets incubated20 min with picotamide(26.8*4.6 ‘A in responseto U46619, 33.6*19.5 0/0in responseto AA, 35.2*17.97. in responseto collagen). Changes in intracellular calcium concentration

Patterns of the changes in intraplateletcalcium concentrationin response to U46619 and AA are shown in Fig. 2, either in picotamide-treatedplatelets subsequently gelfiltered, or in GFP treated with picotamidebefore the agonist addition. If the activation was achieved with U46619, picotamide had the same inhibitory effect both when it was added before the agonist and when it was washed out (250 f 132 AO/O and 216 + 94 AYOrespectivelyversus 697 f 357 AOAof untreatedplatelets). Similar results were obtained when the platelets were activated with AA. In fact, the reduction of AOIO of intracellular calcium concentration was similar either when picotamidewas added before the agonistor it was washed out (152 f 54 AYOand 131 ~ 67 AYo respectively versus 595+ 235 AO/Oof untreatedplatelets). Serotonin release in digitonin-permeabilized platelets stimulated with U46619.

Fig. 3a reports a representative pattern of the serotonin release in response to U46619 both in picotamide-treatedgel-filtered platelets and in untreated platelets. When picotamide was added 20 min before the agonist,the responseto U46619 was lower than the one obtained in untreatedplatelets.

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M for20 minat 37”C)andthengel-filtered(B)andin GFPincubatedwithpicotamide 20 min before the agonist addition (C) in responseto 250 nM U46619and to 75 MM

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Fig. 3b reports a representativepatternof the serotonin release in platelets pretreated with picotamideand then gel-filtered,in responseboth to U46619and to thrombin. The results show an evident inhibition of the responseto U46619 in comparison with the responseto thrombin.

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FIG. 3 Panel a: a representativepattern, of five experimentsperformed, of the serotonin release in digitonin-permeabilizedplatelets in responseto 500 nM U46619 either in picotamide-treated platelets (10-4M for 20 min at 37°C) or in untreated platelets. Control:unstimulatedplatelets. Panel b: a representative pattern, of five experimentsperformed, of the serotonin release in digitonin-permeabilizedplateletspretreatedwith picotamide(10+ M for 20 min at 37°C) before the permeabilizationin responseto 500 nM U46619or 0.2 U/ml thrombin.Control:unstimulatedplatelets. Varying concentrationsof calciumwere usedto assessthe functional integrity of the platelet preparation; in fact, at lowestconcentrationsof calciumthe serotoninrelease is minimalalso in responseto the agonists,whileat highconcentrationsof calciumthe platelets are able to releaseserotonineven in the absenceof the agonist, indicating thus the unpermeabilizedcell rate.

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DISCUSSION The results of the present study show that picotamideeffectwas still present even after it had been washed out from external medium by gel-filtration. In fact, platelet aggregation and serotonin release in responseeither to U46619, a synthetic agonist of the TxA2 receptor, or arachidonicacid or collagen,showed the same inhibition rate either if picotamide was added prior to or after gel-filtration.Similar results have been observed as regards the changes in intracellularcalcium concentration after U46619 and arachidonic acid stimulation. It might be excluded that picotarnideeffect is consequentupon the cytosolic storage of the drug as demonstrated by experiments on digitonin-permeabilized platelets in which picotamide was washed out before the permeabilization. In fact, in platelets pretreated with picotamide and then gel-filtered the pattern of serotonin release in response to U46619 was similar to that obtained in inactivated platelets, while an evident release was achievedonly after stimulationwith thrombin.The same inhibitory effect was observed in platelets where the drug was added after permeabilization, in response to U46619. The fact that the picotamide inhibitory effect on platelets activated with the synthetic agonist of the TXAZreceptorU46619,was still presentafter it was washed out from the external medium, supportsthe hypothesispreviouslysuggestedby Modesti et al. (4,6) that picotamide has a stable interactionwith the thromboxanereceptor. The internalization of TxA2 receptors in platelets following Iigand binding has been suggested by Modesti et al. for both the TxA2receptor antagonistONO11120 (6) and picotamide (4). Moreover, internalizationof TxA2 receptorshas been reported in K562 human leukemia cells following binding of U46619 (15), as well as in liver endothelial cells through binding of TxA2 during cold preservation (16). Our data are not in disagreement with the hypothesis of the internalizationof picotamide/TxAz receptor complexes (4). However, the evidence that the antagonistic effect of picotamide persists even after washing out of the drug from the medium, similarly to what has been shown for other specific TxA2 receptor antagonists (17), strongly substantiates the idea that the long duration of the picotamide effect on platelets depends on its stable non reversible binding to TXAZ receptors rather than on its intracytosolic storage. REFERENCES 1 GRESELE, P., DECKMYN, H., ARNOUT, J., NENCI, G.G. and VERMYLEN, J. Characterization of N,N’-bis(3-Picolyl)-4-Methoxy-lsophthalamide(Picotamide) as a dual Thromboxane Synthase lnhibitor/Thromboxane A2 Receptor Antagonist in Human Platelets.Thromb Haemostas61, 479-484, 1989. 2 CATTANEO,M., TENCONI, P.M., LECCHI,A. and MANNUCCI,P.M. In Vitro Effects of Picotamide on Human Platelet Aggregation, the Release Reaction and Thromboxane62 Production.Thromb Res 62, 717-724, 1991. 3 VIOLI, F., GHISELLI, A., IULIANO, L., PRATICO’, D., ALESSANDRI, C. and BALSANO, F. Inhibition by Picotamide of Thromboxane Production In Vitro and Ex Vivo. Eur J Clin Pharmacol33, 599-602, 1988.

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4 MODESTI, P.A., CECIONI, l., COLELLA,A., COSTOLI,A., PANICCIA,R. and NERI SERNERI, G.G. Binding kinetics and antiplatelet activities of picotamide, a thromboxaneAZ receptorantagonist.Br J Pharmacol 112,81-86, 1994. 5 MODESTI, P.A., COLELLA, A., ABBATE, R., GENSINI, G.F. and NERI SERNERI, G.G. Competitive Inhibition by Picotamide of Platelet Thromboxane A2 Receptor Binding. Eur J Pharmacol 169,85-93, 1989.

6 MODESTI, P.A., COLELLA,A., CECIONI,l., GENSINI,G.F., ABBATE, R. and NERI SERNERI, G.G. Acute Reduction of TxA2 platelet binding sites after in vivo administrationof a TxA2 receptorinhibitor.Br J Clin Pharmacol31, 439-443, 1991. 7 PULCINELLI,F.M., PIGNATELLI,P., RIONDINO,S., PARISI, S., CASTIGLIONI,C. and GAZZANIGA, P.P. Effect of picotamide on the calcium mobilization and phospholipaseC activation in human platelets.Thromb Res 74, 453-461, 1994. 8 ASTER, R.H. and JANDEL, J.H. Platelet Sequestrationin Man. 1.Methods. J Ciin Invest 43, 834-855, 1964. 9 BORN, G.V.R. The aggregationof blood plateletsby adenosinediphosphateand its reversal. Nature 194,405-407, 1962. 10 COSTA, J.J. and MURPHY, D.L. Platelet5-HT uptake and release stopped rapidly by formaldehyde.Nature 255, 405-407, 1975. 11 GRYNKIEWICZ, G., POENIE, M. and TSIEN, R.Y. A New Generation of Ca2+ Indicators with Greatly Improved Fluorescence Properties. J Biol Chem 260, 34403445, 1985. 12 DANIEL,J.L., SELAK, M,A., PURDON,A.D. and SALGANICOFF,L. Methodsfor the study of the role of calcium in platelet function. In: Methods for studying p/ate/ets and rnegakaryocytes. Colman, R.W. and Smith, J.B (eds.), pp. 185-215, Alan R. Liss Inc., New York (1987). 13 PERRIN, D.D. and SAYCE, I.G. Computercalculationof equilibrium concentrations in mixtures of metal ions and completing species.Talanta 14,833-842, 1967. 14 HASLAM, R.J. and DAVIDSON, M.M.L. Guanine nucleotides decrease the free [Ca2+]required for secretionof serotoninfrom permeabilizedblood platelets. Evidence of a role for a GTP-bindingprotein in plateletactivation.FEBS Iett 174,90-95, 1984. 15 DORN, G.W. Mechanism for homologous downregulation of thromboxane A2 receptors in cultured human chronic myelogenousleukemia (K562) cells. J Pharmacol Exp Ther 259, 228-234, 1991 16 ISHIGURO, S., ARII, S., MONDEN,K., FUJITA,S., NAKAMURA,T., NIWANO, M., HARADA, T., USHIKUBI, F., NARUMIYA, S. and IMAMURA, M. Involvement of thromboxane A2-thromboxane A2 receptor system of the hepatic sinusoid in pathogenesis of cold preservation/reperf usion injury in the rat liver graft. Transplantation 59, 957-961, 1995.

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17 ARMSTRONG,R.A., HUMPHREY,P,PA. and LUMLEY,P. Reductionin the number of thromboxane receptors on human platelets after exposure to GR32191. Br J Pharmacol 110,548-552, 1993.