- Email: [email protected]
CURRENT STATUS OF PLATELET NO SYNTHASES
. Thrombosis Research, Vol. 87, No. 1, pp. 51-55,1997 Copyright O 1997 Elsevier Science Ltd Printed in the USA. All rights r=semed C049-384V97 $17.00+ .Ix3
Pergamon
PII SO049-3848(97)O01 O3-5
MINI-REVIEW CURRENT STATUS OF PLATELET NO SYNTHASES Berkels R.’, Stockklauser K.2,RosenP.2,RosenR.’ ‘ Institutfur Pharrnakologie,Universitatzu Koln, Germany 2DiabetesForschungsinstitut,Universitatzu Dusseldorf,Germany (Received 7 October 1996 by EditorJ. Sturzebecher; revised/amepted 25 April 1997)
Nitric oxide is an importantphysiologicalmessengerwith variousbiologicalproperties.It serves as a neuronal messenger in the brain [1] and the non-adrenergicnon-cholinergicnerve system [2]. Macrophages contain art inducible NO synthase isoform which is activated during immunologicalresponses [3], The continuousgenerationof NO by the vascular endotheliumis crucial for the regulation of blood pressure and blood flow [4]. NO is released by several isoformsof NO synthasesfrom the guanidinomoiety of L-arginine,yielding NO and citrulline [5]. Cofactors(NADPH, FMN, FAD, 02, tetrahydrobiopterine,calmodulin)are needed for this conversion via intermediates (hydroxy-L-arginine)[6]. NO is a lipophilic, highly diffusible, moleculewith a short half life. It activatesthe guanylate cyclasethus elevatingcGMP levels [71 which results in severalintracellularreactionssuch as activationof protein kinases and lowering of intracellularcalcium [8]. Endothelium-derivedNO is importantfor the preventionof platelet adhesion and aggregation [4]. During the last few years there has been accumulatingevidence that plateletsthemselvesexhibitan L-arginine/NOpathwaywhich may act as a negativefeedback mechanismto inhibit excessiveactivation/aggregation.Sinceplatelet L-arginine/NOmetabolism is not very well known this review summarises data concerning platelet NO synthases and plateletNO releasein normaland pathologicalconditions. PLATELETNO SYNTHASES Three different isoforms of NO synthase are known, the constitutive calcium/calmodulindependentbrain type (Isoform 1, bNOS) [9], the induciblecalciumindependenttype (Isoform2,
Key words:nitric oxide,platelets,plateletNO synthases,plateletNO release Abbreviations: EMBL, European Molecular Biology Laboratory, ecNOS, endothelial constitutiveNO synthase;iNOS, inducibleNO synthase;NOS, NO synthases Correspondingauthor:Dr. R. Berkels,Institutfti Pharmakologie,Gleueler Str. 24, 50931 Koln, Germany,Tel: +49 221 4783405;FAX: +49 2214785022
51
52
PLATELETNO SYNTHASES
Vol.
87, No. 1
iNOS) [10] and the constitutive calciurdcalmodulin-dependentendothelial type (Isoform 3, ecNOS) [11]. To characteriseNOS isoformsin plateletsdifferentmethodicalapproacheswere used. Recently, Mehta et al. presented evidence (using reverse transcriptionpolymerase chain reaction) for a ecNOS mRNA as well as an inducibleisoform of human platelet NOS [12]. Our group could confirm these results in porcine platelets evidencing an iNOS and ecNOS like mRNA which showed = 85 % homology (EMBL-Accession numbers: sus scrofa iNOS: EMBL-name: SSINDUCNO;EMBL-AccessionNO X 98196; sus scrofa cNOS: EMBL-name:SSCONSTNO; EMBL-Accession NO X 98197) of the platelet cDNA fragments with that of human macrophagesand endothelialcells (matchingto the completecDNA sequences)[13]. On the protein level, ecNOS was evidencedas an immunoreactiveprotein in Western blots of cytosolicfractions [14] or membrane fractions [15] of human platelets. In porcine platelets we could confirm these findings, although immunoreactive protein was only present in the membrane fraction [13], as it was previously described by Sase and Michel [15]. Moreover, using a fluorescencelabeled antibody against ecNOS we could show a prominent cell specific fluorescenceof plateletsin a porcinewhole blood smear [13].Muruganandamet al, were able to isolate a NOS isoform from human platelets by sequentialaffinity chromatographywhich was calcium/calmodulin-dependentand immunoreactiveto ecNOS antibodies [14]. It seems that the plateletNOS is similarto the calciudcalmodulin-dependentecNOS and may be activatedduring plateletactivation/aggregation. We could show that the molecular weight of the platelet NOS protein is = 140 kDa which is consistentwith the results of other authors (= 150 kDa Muruganandamand Mutus [14]; = 130 kDa Methaet al. [12]). NO RELEASEFROM PLATELETS Radomskiet al. were the first to describe an L-arginine/NOpathway in platelets [16, 17]. They were able to show NO release from platelet cytosol (spectroscopicdetermination)which was increased by L-arginine and inhibited by specific NOS inhibitors. Collagen-inducedplatelet aggregationwas accompaniedby an increaseof intracellularcGMP. L-ar@& inhibitedplatelet aggregationand increasedcGMP levels.Noris et al. confirmedthese findingsusing an assay to measure the conversionof labeled L-ar@IIe to L-citrulline[18]. They were also able to show NO release from platelets stimulated with collagen. Without stimulation no nitric oxide liberation was detected. Using a porphyrinic microsensor, Malinski et al. directly measured plateletNO release in whole blood and plateletsuspensionsduringcollagen-inducedaggregation which was blockedby an NOS inhibitorand potentiatedby L-arginine[19]. Basal release of NO was not detectable.Using an electrochemicalNO detectionassay, these results were confirmed by Lantoine et al. [20]. These authors showedNO release from platelets only after stimulation with collagen which is consistentwith histologicalfindings (NADPH diaphorase staining)that an activeNOS is present only in stimulatedplatelets [21].NADPH diaphorasestaininguses the abilityof the activeNOS reductasedomainto convertnitrobluetetrazoliumto formazanwhich is shown histologicallyas a blue colour. Valid data to induce NO release is only available for collagen stimulatedplatelet activatiotiaggregationalthoughin ADP-inducedaggregationthe Larginine/NO pathway may also be involved since L-arginineinhibited ADP-induced platelet aggregation,but there are as yet no valid data availableconcerningNO release [16]. All these data confirmthe presence of a calciumdependentisoformsimilarto the ecNOS. However, only Zhou et al. found significantNO release from resting platelets (spectroscopicdetermination),
Vol.87, No. 1
PLATELETNO SYNTHASES
53
stimulationwith collagenresultedin an increasedNO liberation[22]which confirmsthe findings of Noris et al and Malinski et al [18, 19]. Since the spectroscopicNO measurement is less sensitive and less specific than the electrochemicalNO measurement more evidence for NO release from resting platelets must be obtained. Interestingly,Chen and Metha were able to induce iNOS activity in platelets by incubationwith cytokines [23]. In calcium-ffee buffer, Largininewas converted to citrullineand NO indicatingan induciblecalcium independentNOS isoform althoughthis is surprisingsince plateletshave little capacity for protein biosynthesis.It seemsthat the enzymeis rdreadypresentbut in an inactivestate. During platelet activatiordaggregationintracellularcalcium levels are elevated [24] which may activate platelet NOS thus releasing significant amounts of NO which can act as a negative feedbackmechanismto suppressexceedingactivation/aggregaticm. PLATELETNO SYNTHESISIN PATHOLOGICALCONDITIONS There is accumulatingevidencethat in certain pathologicalconditionsplatelet NO synthesisis altered. Patientswith chronic renal failure are known to have a prolongedbleeding time which may be due to increased L-argininelevels in platelets and an increased NO generation [18]. Patientsundergoinghaemodialysisshowed a loss of sensitivityto nitric oxide possibly due to a desensitizedguanylatecyclasewhich may have been activatedexcessivelywithin the circulation, thus reflectingzmincreasedNO synthesisin platelets [25]. However,the desensitizedguanylate cyclase in platelets may have been influenced by endothelium-derivedNO. The decreased platelet flmctioncaused by high density lipoproteinis due to an increased nitric oxide synthase activity which has been shown in vitro [26]. In patients with a schizophrenic disorder an increasedNOS activitywas also established[27]. DecreasedNO synthesis,possibly due to a platelet hyporesponsivenessto collagen stimulation, mightoccur in patientswith migraine[28]and in patientswith essentialhypertension[29].These examplesindicate that, in the future, it might be beneficialto either use isoform specific NOS inhibitorsto prevent an increasedNOS activitywhich are presentlynot available,or possiblyto supporta decreasedplateletNO productionby NO donors or L-argininesupplementationwhich might inhibit platelet aggregation [30]. Another possibility could be the inhibition of cGMP breakdown by specific cGMP phosphodiesterase inhibitors. More isoform specific NOS inhibitors, especially iNOS inhibitors would be beneficial for therapeutic use, in general. Moreover,it must be kept in mind that drugs given in vivo also act on the endothelialNOS, thus they may exert a concertedreactionon plateletsand the endotheIium.However,the mechanisms by which NO synthases in platelets are regulated and modulated remain to be investigated further. REFERENCES 1. KNOWLES, R. G., PALACIOS, M., PALMER, R. M. and MONCADA, S. Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulationof the solubleguanylatecyclase.ProcNatl Acad Sci USA 86, 5159-5162, 1989. 2. SANDERS, K.M. and WARD, S.M. Nitric oxide as a mediator of nonadrenergic noncholinergicneurotransmission.Am J Physiol262, G379 - G383, 1992. 3. STUEHR,D. J., CHO, H.J., KWON,N.S., WEISE, M.F. and NATHAN, C.F. Purificationand characterizationof the cytokine-inducedmicrophage nitric oxide synthase:an FAD- and FMNcontainingflavoprotein.ProcNatl Acad Sci USA 88, 7773-7777, 1991.
54
PLATELETNO SYNTHASES
vol. 87, No. 1
4. MONCADA, S., PALMER R.M.J. and HIGGS, E.A. Nitric oxide: physiology, pathophysiologyand pharmacology.PharmacolRev 43, 109-142, 1991. 5. PALMER, R.M., ASHTON, D.S. and MONCADA, S. Vascular endothelialcells synthesize nitric oxidefrom L-arginine.Nature 333, 664-666, 1988. 6. FORSTERMANN, U., CLOSS, E I., POLLOCK, J.S., NAKANE, M., SCHWARZ, P., GATH, I. and KLEINERT, H. Nitric oxide synthaseisoenzymes/ characterisation,purification, molecularcloningand functions.Hypertension23, 1121-1131, 1994. 7. BOHME, E., GROSSMANN, G., HERZ, J., MULSCH, A., SPIES, C. and SCHULTZ, G. Regulationof cyclic GMP formationby solubleguanylatecyclasestimulationby NO-containing compounds.Adv CyclicNucleotProteinPhosphorylRes 17, 259-266, 1994. 8. NAKASHIMA, S., TOHMATSU, T., HATTORI, H., OKANO, Y. and NOZAWA, Y. Inhibitoryaction of cGMP on secretion,phosphoinositidehydrolysisand calciummobilisationin thrombin-stimulatedhuman platelets.Biochem.Biophys.Res. Commun.135, 1099-1104, 1986. 9. BREDT, D.S. and SNYDER, S.H. Isolationof nitric oxide synthetase,a calmodulin-requiring enzyme.ProcNatl Acad Sci USA 87, 682-685, 1990. 10. HEVEL, J.M., WHITE, K.A. and MARLETTA, M.A. Purificationof the induciblemurine microphage nitric oxide synthase: identificationas a flavoprotein.J Biol Chem 266, 2278922791, 1991. 11. SESSA, W.C., HARRISON, J.K., BARBER, C.M., ZENG, D., DURIEUX, M.E., D’ANGELO, D.D. and PEACH, M.J. Molecular cloning and expression of a cDNA encoding endothelialcell nitric oxide synthase.J Biol Chem 267, 15274-15276, 1992. 12. MEHTA, J.L., CHEN, L.Y., KONE, C.B., MEHTA, P. and TURNER, P. Identificationof constitutiveand inducibleforms of nitric oxide synthasein humanplatelets.J Lab Clin Med 125, 370-377, 1995. 13. BERKELS, R. BERTSCH, A., ZUTHER, T., DHEIN, S., STOCKKLAUSER,K., ROSEN, P., and ROSEN, R. Evidence for a NO synthasein porcine platelets which is stimulatedduring activation/aggregation.Eur J Haematol,in press, 1997. 14. MURUGANANDAM,A. and MUTUS, B. Isolationof nitric oxide synthasefrom platelets. BiochimBiophysActa 1200, 1-6, 1994. 15. SASE, K. and MICHEL, T. Expressionof constitutiveendothelialnitric oxide synthase in humanbloodplatelets.Life Sci 57,2049-2055, 1995. 16. RADOMSKI,M.W., PALMER, R. and MONCADA,S. An L-arginine/nitricoxide pathway in humanplateletsregulatesaggregation.ProcNatl Acad Sci USA 87, 5193-5197, 1990. 17. RADOMSKI, M. W., PALMER, R.M.J. and MONCADA, S. Characterisationof the Largininenitric oxidepathwayin humanplatelets.Br J Pharmacol101, 325-328, 1990. 18. NORIS, M., BENIGNI, A., BOCCARDO, P., AIELLO, S., GASPARI, F., TODESCHINI, M., FIGLIUZZI,M. and REMUZZ, I.G. Enhancednitric oxide synthesisin uremia: Implications for plateletdysfunctionand dialysishypotension.KidneyInt 44,445-450, 1993. 19.MALINSKI,T., RADOMSKI,M.W., TAHA, Z. and MONCADA,S. Direct electrochemical measurementof nitric oxidereleasedfrom humanplatelets.BiochemBiophysRes Commun 194, 960-965, 1993. 20. LANTOINE,F., BRUNET, A., BEDIOUI,F., DEVYNCK,J., and DEVYNCK, M.A. Direct measurement of nitric oxide production in platelets: relationship with cytosdic Ca2+ concentration.BiochemBiophysRes Commun215, 842-848, 1995. 21. BERKELS,R., CORDES,A., MEYER,D., KLAUS, W. and ROSEN, R. Characterisationof an endogenousL-arginine/NOpathwayin platelets.Endothelium3 Suppl.,S18, 1995.
Vol.87, No. 1
PLATELETNO SYNTHASES
55
22. ZHOU, Q., }Ii?J.LERMANN,G.R. and SOLOMONSON, L.P. Nitric oxide release from restinghumanplatelets,ThrombRes 77, 87-96, 1995.
23. CHIIN,L.Y. and METHA, J.L. Furtherevidenceof the presenceof constitutiveand inducible nitric oxidesynthaseisoformsin humanplatelets.J CardiovascPharmaccd27, 154-8, 1996, 24. KROLL, M., SCHAFER,A. Biochemicalmechanismsof plateletactivation.Blood 74, 1185 -95, 1989 25. GORDGE, M,p. and NEILD, G.H, Plateletsfrom patientson haemodialysisshow impaired responsesto nitricoxide. Clin Sci 83, 313-318, 1992. 26. CHEN, L.Y. and MEHTA, J.L. Inhibitory effect of’high-density lipoprotein on platelet firnctionis mediatedby increasein nitric oxide synthaseactivityin platelets.Life Sci, 55, 18151821,1994. 27. DAS, L, KHAN, N.S., PURI, B.K., SOCUUWNA,S.R., DE BELLEROCHE, J. and HIRSCH, S.R. Elevated platelet calcium mobilisationand nitric oxide synthase activity may reflect abnormalitiesin schizophrenicbrain, Biochem.Biophys.Res. Commun. 212, 375-380, 1995. 28. D’ANDREA, G., CANANZI, A.R., PERINI, F., ALECCIj M., ZAMBERLAN, F., HASSELMARK,L. and WELCH, K.M.A. Decreasedcollagen-inducedplatelet aggregationand increasedplatelet argininelevels in migraine:a possiblelink with the NO pathway. Cephalalgia 14,352-356, 1994. 29. CADWAGN,T.M. and BENJAMIN,N. Evidencefor alteredplateletnitric oxide synthesisin essentialhypertension.J Hypertension11,417-420, 1993. 30. ADAMS, M.R., FORSYTH C.J., JESSUP, W., ROBINSON, J. and CELERMAJER, D.S. Oral L-arginine inhibits platelet aggregation but does not enhance endothelium-dependent dilationin healthyyoungmen. J Am Coil Cardiol.26: 1054-1061, 1995.
Pergamon
PII SO049-3848(97)O01 O3-5
MINI-REVIEW CURRENT STATUS OF PLATELET NO SYNTHASES Berkels R.’, Stockklauser K.2,RosenP.2,RosenR.’ ‘ Institutfur Pharrnakologie,Universitatzu Koln, Germany 2DiabetesForschungsinstitut,Universitatzu Dusseldorf,Germany (Received 7 October 1996 by EditorJ. Sturzebecher; revised/amepted 25 April 1997)
Nitric oxide is an importantphysiologicalmessengerwith variousbiologicalproperties.It serves as a neuronal messenger in the brain [1] and the non-adrenergicnon-cholinergicnerve system [2]. Macrophages contain art inducible NO synthase isoform which is activated during immunologicalresponses [3], The continuousgenerationof NO by the vascular endotheliumis crucial for the regulation of blood pressure and blood flow [4]. NO is released by several isoformsof NO synthasesfrom the guanidinomoiety of L-arginine,yielding NO and citrulline [5]. Cofactors(NADPH, FMN, FAD, 02, tetrahydrobiopterine,calmodulin)are needed for this conversion via intermediates (hydroxy-L-arginine)[6]. NO is a lipophilic, highly diffusible, moleculewith a short half life. It activatesthe guanylate cyclasethus elevatingcGMP levels [71 which results in severalintracellularreactionssuch as activationof protein kinases and lowering of intracellularcalcium [8]. Endothelium-derivedNO is importantfor the preventionof platelet adhesion and aggregation [4]. During the last few years there has been accumulatingevidence that plateletsthemselvesexhibitan L-arginine/NOpathwaywhich may act as a negativefeedback mechanismto inhibit excessiveactivation/aggregation.Sinceplatelet L-arginine/NOmetabolism is not very well known this review summarises data concerning platelet NO synthases and plateletNO releasein normaland pathologicalconditions. PLATELETNO SYNTHASES Three different isoforms of NO synthase are known, the constitutive calcium/calmodulindependentbrain type (Isoform 1, bNOS) [9], the induciblecalciumindependenttype (Isoform2,
Key words:nitric oxide,platelets,plateletNO synthases,plateletNO release Abbreviations: EMBL, European Molecular Biology Laboratory, ecNOS, endothelial constitutiveNO synthase;iNOS, inducibleNO synthase;NOS, NO synthases Correspondingauthor:Dr. R. Berkels,Institutfti Pharmakologie,Gleueler Str. 24, 50931 Koln, Germany,Tel: +49 221 4783405;FAX: +49 2214785022
51
52
PLATELETNO SYNTHASES
Vol.
87, No. 1
iNOS) [10] and the constitutive calciurdcalmodulin-dependentendothelial type (Isoform 3, ecNOS) [11]. To characteriseNOS isoformsin plateletsdifferentmethodicalapproacheswere used. Recently, Mehta et al. presented evidence (using reverse transcriptionpolymerase chain reaction) for a ecNOS mRNA as well as an inducibleisoform of human platelet NOS [12]. Our group could confirm these results in porcine platelets evidencing an iNOS and ecNOS like mRNA which showed = 85 % homology (EMBL-Accession numbers: sus scrofa iNOS: EMBL-name: SSINDUCNO;EMBL-AccessionNO X 98196; sus scrofa cNOS: EMBL-name:SSCONSTNO; EMBL-Accession NO X 98197) of the platelet cDNA fragments with that of human macrophagesand endothelialcells (matchingto the completecDNA sequences)[13]. On the protein level, ecNOS was evidencedas an immunoreactiveprotein in Western blots of cytosolicfractions [14] or membrane fractions [15] of human platelets. In porcine platelets we could confirm these findings, although immunoreactive protein was only present in the membrane fraction [13], as it was previously described by Sase and Michel [15]. Moreover, using a fluorescencelabeled antibody against ecNOS we could show a prominent cell specific fluorescenceof plateletsin a porcinewhole blood smear [13].Muruganandamet al, were able to isolate a NOS isoform from human platelets by sequentialaffinity chromatographywhich was calcium/calmodulin-dependentand immunoreactiveto ecNOS antibodies [14]. It seems that the plateletNOS is similarto the calciudcalmodulin-dependentecNOS and may be activatedduring plateletactivation/aggregation. We could show that the molecular weight of the platelet NOS protein is = 140 kDa which is consistentwith the results of other authors (= 150 kDa Muruganandamand Mutus [14]; = 130 kDa Methaet al. [12]). NO RELEASEFROM PLATELETS Radomskiet al. were the first to describe an L-arginine/NOpathway in platelets [16, 17]. They were able to show NO release from platelet cytosol (spectroscopicdetermination)which was increased by L-arginine and inhibited by specific NOS inhibitors. Collagen-inducedplatelet aggregationwas accompaniedby an increaseof intracellularcGMP. L-ar@& inhibitedplatelet aggregationand increasedcGMP levels.Noris et al. confirmedthese findingsusing an assay to measure the conversionof labeled L-ar@IIe to L-citrulline[18]. They were also able to show NO release from platelets stimulated with collagen. Without stimulation no nitric oxide liberation was detected. Using a porphyrinic microsensor, Malinski et al. directly measured plateletNO release in whole blood and plateletsuspensionsduringcollagen-inducedaggregation which was blockedby an NOS inhibitorand potentiatedby L-arginine[19]. Basal release of NO was not detectable.Using an electrochemicalNO detectionassay, these results were confirmed by Lantoine et al. [20]. These authors showedNO release from platelets only after stimulation with collagen which is consistentwith histologicalfindings (NADPH diaphorase staining)that an activeNOS is present only in stimulatedplatelets [21].NADPH diaphorasestaininguses the abilityof the activeNOS reductasedomainto convertnitrobluetetrazoliumto formazanwhich is shown histologicallyas a blue colour. Valid data to induce NO release is only available for collagen stimulatedplatelet activatiotiaggregationalthoughin ADP-inducedaggregationthe Larginine/NO pathway may also be involved since L-arginineinhibited ADP-induced platelet aggregation,but there are as yet no valid data availableconcerningNO release [16]. All these data confirmthe presence of a calciumdependentisoformsimilarto the ecNOS. However, only Zhou et al. found significantNO release from resting platelets (spectroscopicdetermination),
Vol.87, No. 1
PLATELETNO SYNTHASES
53
stimulationwith collagenresultedin an increasedNO liberation[22]which confirmsthe findings of Noris et al and Malinski et al [18, 19]. Since the spectroscopicNO measurement is less sensitive and less specific than the electrochemicalNO measurement more evidence for NO release from resting platelets must be obtained. Interestingly,Chen and Metha were able to induce iNOS activity in platelets by incubationwith cytokines [23]. In calcium-ffee buffer, Largininewas converted to citrullineand NO indicatingan induciblecalcium independentNOS isoform althoughthis is surprisingsince plateletshave little capacity for protein biosynthesis.It seemsthat the enzymeis rdreadypresentbut in an inactivestate. During platelet activatiordaggregationintracellularcalcium levels are elevated [24] which may activate platelet NOS thus releasing significant amounts of NO which can act as a negative feedbackmechanismto suppressexceedingactivation/aggregaticm. PLATELETNO SYNTHESISIN PATHOLOGICALCONDITIONS There is accumulatingevidencethat in certain pathologicalconditionsplatelet NO synthesisis altered. Patientswith chronic renal failure are known to have a prolongedbleeding time which may be due to increased L-argininelevels in platelets and an increased NO generation [18]. Patientsundergoinghaemodialysisshowed a loss of sensitivityto nitric oxide possibly due to a desensitizedguanylatecyclasewhich may have been activatedexcessivelywithin the circulation, thus reflectingzmincreasedNO synthesisin platelets [25]. However,the desensitizedguanylate cyclase in platelets may have been influenced by endothelium-derivedNO. The decreased platelet flmctioncaused by high density lipoproteinis due to an increased nitric oxide synthase activity which has been shown in vitro [26]. In patients with a schizophrenic disorder an increasedNOS activitywas also established[27]. DecreasedNO synthesis,possibly due to a platelet hyporesponsivenessto collagen stimulation, mightoccur in patientswith migraine[28]and in patientswith essentialhypertension[29].These examplesindicate that, in the future, it might be beneficialto either use isoform specific NOS inhibitorsto prevent an increasedNOS activitywhich are presentlynot available,or possiblyto supporta decreasedplateletNO productionby NO donors or L-argininesupplementationwhich might inhibit platelet aggregation [30]. Another possibility could be the inhibition of cGMP breakdown by specific cGMP phosphodiesterase inhibitors. More isoform specific NOS inhibitors, especially iNOS inhibitors would be beneficial for therapeutic use, in general. Moreover,it must be kept in mind that drugs given in vivo also act on the endothelialNOS, thus they may exert a concertedreactionon plateletsand the endotheIium.However,the mechanisms by which NO synthases in platelets are regulated and modulated remain to be investigated further. REFERENCES 1. KNOWLES, R. G., PALACIOS, M., PALMER, R. M. and MONCADA, S. Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulationof the solubleguanylatecyclase.ProcNatl Acad Sci USA 86, 5159-5162, 1989. 2. SANDERS, K.M. and WARD, S.M. Nitric oxide as a mediator of nonadrenergic noncholinergicneurotransmission.Am J Physiol262, G379 - G383, 1992. 3. STUEHR,D. J., CHO, H.J., KWON,N.S., WEISE, M.F. and NATHAN, C.F. Purificationand characterizationof the cytokine-inducedmicrophage nitric oxide synthase:an FAD- and FMNcontainingflavoprotein.ProcNatl Acad Sci USA 88, 7773-7777, 1991.
54
PLATELETNO SYNTHASES
vol. 87, No. 1
4. MONCADA, S., PALMER R.M.J. and HIGGS, E.A. Nitric oxide: physiology, pathophysiologyand pharmacology.PharmacolRev 43, 109-142, 1991. 5. PALMER, R.M., ASHTON, D.S. and MONCADA, S. Vascular endothelialcells synthesize nitric oxidefrom L-arginine.Nature 333, 664-666, 1988. 6. FORSTERMANN, U., CLOSS, E I., POLLOCK, J.S., NAKANE, M., SCHWARZ, P., GATH, I. and KLEINERT, H. Nitric oxide synthaseisoenzymes/ characterisation,purification, molecularcloningand functions.Hypertension23, 1121-1131, 1994. 7. BOHME, E., GROSSMANN, G., HERZ, J., MULSCH, A., SPIES, C. and SCHULTZ, G. Regulationof cyclic GMP formationby solubleguanylatecyclasestimulationby NO-containing compounds.Adv CyclicNucleotProteinPhosphorylRes 17, 259-266, 1994. 8. NAKASHIMA, S., TOHMATSU, T., HATTORI, H., OKANO, Y. and NOZAWA, Y. Inhibitoryaction of cGMP on secretion,phosphoinositidehydrolysisand calciummobilisationin thrombin-stimulatedhuman platelets.Biochem.Biophys.Res. Commun.135, 1099-1104, 1986. 9. BREDT, D.S. and SNYDER, S.H. Isolationof nitric oxide synthetase,a calmodulin-requiring enzyme.ProcNatl Acad Sci USA 87, 682-685, 1990. 10. HEVEL, J.M., WHITE, K.A. and MARLETTA, M.A. Purificationof the induciblemurine microphage nitric oxide synthase: identificationas a flavoprotein.J Biol Chem 266, 2278922791, 1991. 11. SESSA, W.C., HARRISON, J.K., BARBER, C.M., ZENG, D., DURIEUX, M.E., D’ANGELO, D.D. and PEACH, M.J. Molecular cloning and expression of a cDNA encoding endothelialcell nitric oxide synthase.J Biol Chem 267, 15274-15276, 1992. 12. MEHTA, J.L., CHEN, L.Y., KONE, C.B., MEHTA, P. and TURNER, P. Identificationof constitutiveand inducibleforms of nitric oxide synthasein humanplatelets.J Lab Clin Med 125, 370-377, 1995. 13. BERKELS, R. BERTSCH, A., ZUTHER, T., DHEIN, S., STOCKKLAUSER,K., ROSEN, P., and ROSEN, R. Evidence for a NO synthasein porcine platelets which is stimulatedduring activation/aggregation.Eur J Haematol,in press, 1997. 14. MURUGANANDAM,A. and MUTUS, B. Isolationof nitric oxide synthasefrom platelets. BiochimBiophysActa 1200, 1-6, 1994. 15. SASE, K. and MICHEL, T. Expressionof constitutiveendothelialnitric oxide synthase in humanbloodplatelets.Life Sci 57,2049-2055, 1995. 16. RADOMSKI,M.W., PALMER, R. and MONCADA,S. An L-arginine/nitricoxide pathway in humanplateletsregulatesaggregation.ProcNatl Acad Sci USA 87, 5193-5197, 1990. 17. RADOMSKI, M. W., PALMER, R.M.J. and MONCADA, S. Characterisationof the Largininenitric oxidepathwayin humanplatelets.Br J Pharmacol101, 325-328, 1990. 18. NORIS, M., BENIGNI, A., BOCCARDO, P., AIELLO, S., GASPARI, F., TODESCHINI, M., FIGLIUZZI,M. and REMUZZ, I.G. Enhancednitric oxide synthesisin uremia: Implications for plateletdysfunctionand dialysishypotension.KidneyInt 44,445-450, 1993. 19.MALINSKI,T., RADOMSKI,M.W., TAHA, Z. and MONCADA,S. Direct electrochemical measurementof nitric oxidereleasedfrom humanplatelets.BiochemBiophysRes Commun 194, 960-965, 1993. 20. LANTOINE,F., BRUNET, A., BEDIOUI,F., DEVYNCK,J., and DEVYNCK, M.A. Direct measurement of nitric oxide production in platelets: relationship with cytosdic Ca2+ concentration.BiochemBiophysRes Commun215, 842-848, 1995. 21. BERKELS,R., CORDES,A., MEYER,D., KLAUS, W. and ROSEN, R. Characterisationof an endogenousL-arginine/NOpathwayin platelets.Endothelium3 Suppl.,S18, 1995.
Vol.87, No. 1
PLATELETNO SYNTHASES
55
22. ZHOU, Q., }Ii?J.LERMANN,G.R. and SOLOMONSON, L.P. Nitric oxide release from restinghumanplatelets,ThrombRes 77, 87-96, 1995.
23. CHIIN,L.Y. and METHA, J.L. Furtherevidenceof the presenceof constitutiveand inducible nitric oxidesynthaseisoformsin humanplatelets.J CardiovascPharmaccd27, 154-8, 1996, 24. KROLL, M., SCHAFER,A. Biochemicalmechanismsof plateletactivation.Blood 74, 1185 -95, 1989 25. GORDGE, M,p. and NEILD, G.H, Plateletsfrom patientson haemodialysisshow impaired responsesto nitricoxide. Clin Sci 83, 313-318, 1992. 26. CHEN, L.Y. and MEHTA, J.L. Inhibitory effect of’high-density lipoprotein on platelet firnctionis mediatedby increasein nitric oxide synthaseactivityin platelets.Life Sci, 55, 18151821,1994. 27. DAS, L, KHAN, N.S., PURI, B.K., SOCUUWNA,S.R., DE BELLEROCHE, J. and HIRSCH, S.R. Elevated platelet calcium mobilisationand nitric oxide synthase activity may reflect abnormalitiesin schizophrenicbrain, Biochem.Biophys.Res. Commun. 212, 375-380, 1995. 28. D’ANDREA, G., CANANZI, A.R., PERINI, F., ALECCIj M., ZAMBERLAN, F., HASSELMARK,L. and WELCH, K.M.A. Decreasedcollagen-inducedplatelet aggregationand increasedplatelet argininelevels in migraine:a possiblelink with the NO pathway. Cephalalgia 14,352-356, 1994. 29. CADWAGN,T.M. and BENJAMIN,N. Evidencefor alteredplateletnitric oxide synthesisin essentialhypertension.J Hypertension11,417-420, 1993. 30. ADAMS, M.R., FORSYTH C.J., JESSUP, W., ROBINSON, J. and CELERMAJER, D.S. Oral L-arginine inhibits platelet aggregation but does not enhance endothelium-dependent dilationin healthyyoungmen. J Am Coil Cardiol.26: 1054-1061, 1995.