Nitric Oxide and its Antithrombotic Action in the Cardiovascular System

Current Drug Targets - Cardiovascular & Haematological Disorders, 2005, 5, 65-74

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Nitric Oxide and its Antithrombotic Action in the Cardiovascular System Reichenbach Gustavo1,*, Momi Stefania2 and Gresele Paolo2 1

Department of Chemistry, University of Perugia, Perugia, Italy, 2Department of Internal Medicine, University of Perugia, Perugia Italy Abstract: Nitric oxide (NO) is a small gaseous molecule with an odd number of electrons and is rather soluble in hydrophobic phases. It was once known for its toxicity in the environment and for its applications in meat curing. After 1980 its importance was discovered in many physiological fields such as vascular regulation, neuronal communication, cytotoxic action by macrophages in bacterial infections etc. On the other side NO is involved in toxic shock, DNA damage and many pathological conditions. In 1992 the journal Science designated it as “molecule of the year” and in the last years there has been an explosion of publications on the subject. The publications are concerned with the spectroscopic characterisation of NO derivatives, with the reactivity of NO with Myoglobin, Cytochrome and Hemoglobin and in particular with the chemical activities and biological applications of nitric oxide donors and nitric oxide scavengers. All such researches have produced until now many patents. The most famous products are Viagra and nitroglycerine (Trinitrin). Particular attention is given to the applications of NO to cardiovascular and hematological disorders. To this aim the authors examine the physiologic activities of NO and the mechanism of its antiplatelet, vasodilatory and antiproliferative action. Studies in animals and humans are also reported. Another section examines the drugs that increase the endogenous production of NO and modulate its activities. The last part is dedicated to the novel antithrombotic agent Nitroaspirin. Methods for NO detection will also be examined.

Key Words: Atherosclerosis, Cardiovascular system, Myoglobin, Nitric oxide, Nitric oxide donors, Nitric oxide scavengers, Nitroaspirin, Oxygen radicals, Platelet aggregation, Thrombosis, Vasodilation. INTRODUCTION Nitric Oxide (NO) was once known as a pollutant, but recently it has been discovered that it is very important in animal life and therefore has been studied by chemists, by biologists, by experts in pharmacy and medicine. Some chemists have also studied the interaction of NO and other small molecules with artificial compounds which mimic the behaviour of hemoproteins [1-3]. Due to its importance it has been called in 1998 “The molecule of the year” [4] and a complete volume of “Chemical Review” has been dedicated to it [5] while a Conference has been held in Prague in 2002 on it with more than 300 lectures by experts from all the parts of the world [6] and a journal is completely dedicated to it [7]. In recent years more than 90 patents were presented on it and Furchgott, Ignarro and Murad were awarded the Nobel prize for their discoveries concerning NO [8-10]. NO is a small gaseous molecule rather soluble in hydrophobic phases and is a free radical with an odd number of electrons. It is colorless and paramagnetic. It is unstable and in the presence of oxygen and water oxidizes to nitrites and nitrates. Its half–life in blood is very short (seconds) and cannot be transported away from its source of synthesis. NO is generated in the body through the transformation of arginine in citrulline by three isoforms of NO synthase: endothelial (eNOS), inducible (iNOS) and neural (nNOS). They generate NO continuously to maintain a vasodilator

*Address correspondence to this author at the Department of Chemistry, Università, Perugia, Italy; E-mail: [email protected] 1568-0061/05 $50.00+.00

tone. The problem which is rising is how the molecule can travel through the body since it is very reactive. Stamler et al. [11] proposed that some thiol groups can be nitrosated and are thus capable of transporting NO to distal regions of the body. Moreover some nitrosothiols can transnitrosilate other thiols which can collaborate in the transport of NO group. One tentative explaination has been given by Pawloski et al. [12] and by Gross in a paper very interesting from the scientific and didactic point of view [13]. In their opinion NO reacts with Fe++ of hemoglobin (Hb), then it transfers to the SH group of the same molecule and then to the SH group of AE1, an anion exchanger protein. This molecule or other nitrosothiols transport the NO molecule where it is needed. It is noteworthy that also human myoglobin (Mb), differently from other animal Mb, has a cysteine goup at position 110 [14]. As concerned with such problem, we have studied the interaction of NO, Mb and glutathione: NO reacts quickly with the Fe+++ of Mb in conditions similar to the physiological ones then is tranferred to glutahione [15]. We have used horse muscle Mb, but since the human Mb has one SH group in its proteic part, the mechanism can be different and similar to that proposed for Hb. A study with human Mb would be certainly very interesting. We have also studied the reaction between cytochrome c, cysteine and NO in a similar way, but the reaction cannot be followed since cytochrome c reacts with thiols and oxidises them to disulphides. Nevertheless we have studied the oxidation reaction of cysteine by cytochrome c, obtaining some interesting preliminary results. The reaction has been studied both in the absence and in the presence of oxygen: in the first case the © 2005 Bentham Science Publishers Ltd.

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Current Drug Targets - Cardiovas. & Haemat. Dis., 2005, Vol. 5, No. 1

second order rate constant is 21x10-5 M-1s-1, while in the second one is 17x10-5 M -1s-1. In both cases the rate constant increases on increasing pH and so we think that the reducing agent is the cys-anion. The effect of metallic ions has also been studied , and it has been determined that the effect is positive and can be neutralized by the addition of EDTA [16]. Demoncheaux et al. have recently demonstrated that nitrites release small quantities of NO and so they believe that the vasodilator effect is due directly to nitrites. Small quantities of NO are determined by a method based on luminescence and a special detector (Glaxo Smith Kline) [17]. An interesting dispute has then arised with Tsikas and Frolich who argued that even though the quantities of NO released by nitrites are very small, such small quantities can react with thiols, whose activity as vasodilator is much higher than the activity of free NO [18,19]. NONCARDIOVASCULAR EFFECTS OF NO Nitric Oxide has many effects both positive and deleterious, which have been studied by many groups. Its main effect is to dilate blood vessels and consequently to decrease arterial pressure (vide infra). Nitroglycerine (trinitrin) has been used for many years and even Nobel was very surprised and happy that his discovery could be used to cure angina pectoris. Many groups are involved in the study of such application [20]. Also some blood-sucking insects like Rhodnius Prolixus have “discovered” the proprieties of NO and so when they pick animals or humans they inject NO-carrying proteins which dilate vessels and inhibit platelet aggregation and blood coagulation. Such insects have been widely studied also because they transmit the trypanosome responsible for Chagas’ disease [21,22]. Connected with the vasodilator effect, is the effect of NO on reproductive organs and it has been found that NO and NOS have influence on masculine [23] reproductive organs. In particular the effect of “Viagra” is rather famous and has had a positive influence on the budget of the industry which produced it. NO can have influence on the growth of cancer. For example an overproduction of NO (both endogenous and exogenous) can enhance the effect of radiation on cancer cells [24]. The strategy of starving tumoral cells has been adopted by many researcher. Since tumors are rich of NO, mainly produced by iNOS, which increases the blood flow, the administration of iNOS inhibitors can be a system to contrast such disease. Esumi et al. studied the implication of iNOS in carcinogenesis [25]. Iwase et al. [26] noticed that antioxidants and natural derived compounds have a chemoprotective activity on mouse skin carcinogenesis induced by NO-donors. NO has also an important influence on pulmonary disease. For example T. Schon et al. affirmed that the local production of NO has a role in human tubercolosis [27]. Akaike et al. affirmed that in viral infections there is an overproduction of NO by iNOS. NO reacts with the radical O2 to produce peroxinitrite which contributes to the pathogenesis of virus-induced pneumonia and other diseases [28,29]. In fact the viral infections are reduced by using pharmacologic inhibitors of NOS or in omozigote iNOS deficient mice.

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NO is also important in pregnacy. Recent communications were given by Dzgoeva et al. [30] and by Vedernicov et al. [31,32]. The first authors measured the changes in NO production after pregnacy and after L-arginine injection. The second ones published many papers on the same suject. They compared the situation in pregnant and non-pregnant rats and hypotesized that the relative uterine quiescence during pregnancy is NO-dependent. Other recent papers by the same authors are concerned with the influence of many chemical substances on uterine circulation in pregnants rats. NO can be a useful or toxic substance and can be transformed in nitrosothiols in the aorta and reproductive tissues so that it can be inactivated and stored. Moreover it is well known that nitrosothiols are transporters of NO (vide supra) and so can be used as potential therapeutic agents: they have antiplatelet properties, play an important role in the treatment of asthma and could be used as agents to treat infectious diseases [33]. PRODUCTION AND SCAVENGING OF NO Due to its proprieties, both positives and negatives, important targets are is to produce it or to reduce it if necessary. There are many systems to produce NO, while it is more difficult to scavenge it. Many classes of NO donors are described in Chem. Rev. [34] with 800 references and will be shortly summarised here. a)

There are many organic nitrates and they can release NO through enzymatic or non-enzymatic reactions and have been used for a long time to relieve angina pectoris and other diseases.

b) The organic nitrites produce NO which can react with thiols to give RSNO that can transport it where it is needed. c)

Many metals and metalloorganic complexes can both fix NO to bioregulate its concentration and release it in proper conditions. Two important compounds are Ruand Fe-porphirin derivatives.

d) N-Hydoxy-N-nitrosoammines release NO and particular derivatives have specific targeting effects. They release NO photochemically, enzymatically or chemically in a controlled manner. Moreover the by-products are not carcinogenic. e)

Nitrosothiols are supposed to be important in the storage, transport and delivery of NO. Their stability and decomposition have been repeatedly studied. The spontaneous omolytic decomposition can be excluded due to the strength of the bond RS-NO. So their decomposition can be a complicated (second order) mechanism induced by light, enzymes or metals, especially copper. Since nitosothiols are important NOreleasing molecules, there is a great interest to synthesize novel molecules with good pharmacokinetic properties. In particular nitosothiols connected to a glucose molecule have promising proprieties. RSNO can act as a vasodilator or as antiplatelet agent, both as a NO generator or by itself. He has also other important activities.

Nitric Oxide and its Antithrombotic Action on the Cardiovascular System

f)

Diazeniumdiolates are an important family of chemical compounds, well known for almost 150 years [35]. Their main characteristic is to release NO with a half life ranging from 2 seconds to 200 hours and are used in many medical applications requiring either a rapid production or a gradual release. Moreover important studies are ongoing to obtain derivatives which release NO specifically to a particular site. A system to obtain such aim is to use photochemistry [36].

g) Other important classes of NO donors are the diazetine dioxides, Furoxans, C-nitoso compound, Oxatriazole-5imines etc. h) One other possibility is to connect a NO-donor to known drugs. This can reduce the toxicity of the drug and add other effects connected to the NO-releasing capacity. Apart from the aspirin derivatives (vide infra ), some drugs attach an SNO moiety to non-steroidal drugs. These products produce fewer stomach lesion as compared to the parent drug. Others connect a NOdonor to an alpha-antagonist drug obtaining both vasodilation and alpha antagonist activities. i)

Last but not least some products were synthesized which are enzyme activated so that they can deliver NO in particular regions of the body without affecting other regions. For example Saavedra et al. attached a –CH2OAc group to a diazeniumdiolate [37]. These compounds are inert in neutral acqueous media but release NO after metabolism by esterase. An opposite aim is to scavenge NO, when it is in excess. There are not many substances which can block the molecule selectively. A system widely used is to block it indirectly. Since NO is produced in vivo through the reaction of arginine with NO synthase (vide supra) many researchers have used such system. For example Lopez et al. [38], Griffith et al. [39], Fishlock et al. [40] developed the control of NOS enzyme activity using the localized photochemical release of an isoform-specific NOS inhibitor. A similar strategy was used by Pitzole et al. [41] who synthesized a series of compounds to selectively inhibit iNOS and by Boveris et al. [42] who inhibited mitochondrial NOS activity. A different strategy is used by Herold and coworkers taking into account that NO reacts both with O2- and with derivatives of Mb and Hb. The first reaction is potentially dangerous because the formed peroxinitrites can damage DNA, can nitrate aromatic compounds etc. The second reaction is useful because it can reduce the half-life of NO and reduce its concentration. In particular MbFeO2 reacts with NO to give MbOONO which decades quickly to give nitrates and no peroxinitrites. A similar reaction but faster is produced by the analogous Hb derivative [43]. A similar approach has been described by Witting and coworkers [14] and by Flogel et al. [44], and by Zheng and Birke with reference to glutahionylcobalamin [45]. Herold and coworkers are also studying the influence of CO2 in such reactions, since CO2 reduces the lifetime of peroxinitrites [46]. Connected with the behaviour of Mb, two recent papers have reported impressive results. One affirms that mice with-


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out Mb can have a normal life and even reproduce themselves [47]. This fact rise important questions on the importance of Mb in the metabolism of O2, whose transport can have alternative mechanisms. The other affirms that Mb is rather similar in many animals (horses, sperm whales, rats) but only the human Mb has one SH group (a cysteine in position 110) and so can have a different behaviour from other myoglobines [14]. This aspect could be very important in pharmacologic studies, since the biochemistry of humans could be in some aspects different from that of other animals (It would be nice if the evolution of man, so different from that of other animals, is due to such cysteine in position 110 !). DETECTION OF NO AND ITS DERIVATIVES Many systems have been developed to such goal. The classical method is the colorimetric and fluorimetric Griess system in which NO2- reacts with alpha-naphtil-ammine in an acid ambient to produce an azocompound colored in red. It requires a preliminary displacement of NO+ with HgCl 2 or CuCl2 [48-50]. An instrument based on the Griess reaction is produced by Sigma and by ENO-20 Eicom (Kyoto, Japan). Other methods are based on ESR [51,52] and on chemiluminescence [53]. Other sensors have been produced for example by World Precision Instruments, by Innovatives Instruments Inc., by Assay Design Inc., by Perkin Elmer etc. The system prepared by our group can be used to determine NO bound to RSNOs. It uses a specific solid-state amperometric sensor and allows the determination of NO released from RSNO, with a sensitivity of the order of nanomolar concentrations [54]. PHYSIOLOGIC ACTIVITIES Nitric oxide (NO) exerts several biologic activities in the cardiovascular system in addition to its well known role as a regulator of vascular homeostasis and as a smooth muscle cell relaxant [55]. NO is able to inhibit platelet adhesion and aggregation induced by various stimuli and it has been found to induce disaggregation of preaggregated platelets [56, 57]; it prevents the adhesion of neutrophils to altered endothelium [58]; it inhibits smooth muscle cell migration and proliferation [59] thus preventing the vascular remodelling phenomena that are at the basis of atherogenesis and restenosis [60- 62]. In addition, NO plays a central role in the regulation of other pathophysiological processes involving blood vessels such as inflammation. In inflammation, NO is involved in host defence by favouring vasodilation, edema and cytotoxicity. Conversely, the production of NO by endothelial cells may serve an anti-inflammatory function by decreasing inflammation, cell rolling, adhesion and extravasation [63]. PRODUCTION OF NO WITHIN THE CARDIOVASCULAR SYSTEM The NO synthesizing enzyme (NOS) exists in three isoforms that share about 50% amino acid homology and are encoded by different genes localized in different chromosomes. The three isoforms are the endothelial

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constitutive isoform (ecNOS or NOS III), the neuronal isoform (nNOS or NOS I) and the inducible isoform (iNOS or NOS II) [64, 65]. iNOS expression is regulated by chemotactic and inflammatory mediators and produces large amounts of NO. The NOS isoforms present within the cardiovascular system are eNOS and iNOS. ++

Endothelial cells express eNOS, that is sensitive to Ca , as well as iNOS , that is Ca++ insensitive. The largest bulk of NO within the cardiovascular system is produced by NOS III of endothelial cells. One of the main stimuli that activate the biosynthesis of NO by endothelium is blood flow. The shear stress exerted by pulsatile flow induces NO-release by a Ca++-mediated and ATP independent mechanism [66]. Shear stress causes G-protein-mediated increases in Ca++ influx as well as phospholipase C (PLC) mediated hydrolysis of phosphatidyl inositol bisphosphate yielding inositol triphosphate (IP3) which releases calcium from intracellular stores. The resulting increase in intracellular Ca++ activates the calcium dependent eNOS to produce NO [67]. The chronic production of NO by inducible nitric oxide synthase (iNOS) in heart failure exerts deleterious effects on ventricular contractility and circulatory function [68]. Recent data from our group have demonstrated that high shear stress induces the activation of eNOS also within human blood platelets and that the resulting NO inhibits, by a negative feed-back mechanism, shear stress-induced platelet activation [69]. NO is synthetized also by other cells in the cardiovascular tree, such as platelets (eNOS, iNOS) [70], smooth muscle cells (iNOS), macrophages (iNOS) [71], neutrophils (iNOS) [72] and cardiomyocytes (eNOS) [73]. Intimal SMCs are the main iNOS expressing cell types in the injured artery. Intimal production of NO via the inducible pathway may be important for the restoration of vascular homeostasis after injury [74]. NO is produced by macrophages in response to stimuli such as interferon-gamma with the participation of several second messengers such as JAK2, MEK, Erk 1/Erk 2 and Stat 1 alpha and it regulates the inflammatory response [75]. CELLULAR MECHANISMS OF THE ANTIPLATELET, VASODILATORY AND ANTIPROLIFERATIVE ACTION OF NO Most of the NO effects are mediated by the activation of a soluble guanylyl cyclase and the subsequent rise in intracellular cyclic guanosine 3’-5’ monophosphate (cGMP). Soluble guanylyl cyclase is an ubiquitous cytoplasmic, heme-containing enzyme that has a high affinity for NO; when NO, which can freely cross cell-membranes, reacts with the iron-containing heme moiety of the enzyme, this is activated and converts GTP in cGMP. Cyclic GMP, in turn, activates a cGMP-dependent protein kinase G that phosphorylates several intracellular molecules which favour the reuptake of calcium in intracellular stores, increase calcium extrusion from cells and inhibit calcium influx: all these actions together lead to the decrease of intracellular free Ca++ and turn-off cell activation. The in vivo importance of this mechanism in

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controlling platelet activation is confirmed by the strongly thrombogenic phenotype of mice knockouts for the gene encoding cGKI [76]. cGMP is subsequently scavenged by a variety of phosphodiesterases [77]. NO AND PLATELETS PRODUCTION)

(EFFECTS

AND

NO inhibits a wide range of platelet functional responses to stimuli [78] and modulates thrombosis in vivo. Platelets and their progenitors contain a constitutive NOS identical to endothelial NOS and produce and release NO upon activation [79]. Platelet aggregation is enhanced after incubation with NOS inhibitors, such as L-NAME, and conversely is inhibited by incubation with the natural substrate of NOS, Larginine [70]. Platelet-derived NO can be directly measured in platelet rich plasma or in washed platelets by a NOsensitive electrode after agonist stimulation [80, 81] and this release is potentiated by L-arginine and reduced by NOS inhibitors, confirming the presence of a functionally active L-arginine/NO pathway in human platelets [79, 82]. Platelet-derived NO inhibits P-selectin and GPIIb/IIIa expression in stimulated platelets [83] and endogenousgenerated NO inhibits thrombin receptor-activating peptideinduced PI3 kinase activity, a very basic signal-transduction event in platelet activation [84]. The antiplatelet effects of NO include the inhibition of 12-lipoxygenase and cyclooxygenase-2 and, to a lower extent, cyclooxygenase-1 [85-87]. Moreover, it was shown that platelet recruitment is inhibited by platelet-derived NO confirming this as an important mechanism limiting arterial thrombosis [88]. THROMBOSIS MODELS AND NO The important role of platelet-derived NO as a natural antithrombotic mechanism has also been demonstrated in vivo by the infusion of a NOS inhibitor that enhances platelet reactivity to various agonists [89]. The in vivo antithrombotic relevance of NO was initially suggested by a series of experiments in which S-nitrososerum-albumin was administered intravenously in dogs with deendothelialized stenosed coronary arteries [90]. In this model the effect of S-nitroso serum albumin, an important in vivo reservoir of NO, was predominantly an antiplatelet effect and showed only a mild vasodilatory action [91]. The effects of endogenous NO upon thrombininduced platelet accumulation in the pulmonary vasculature of the rabbit were also examined [92], and found to be in part attributable to an enhancing effect on endogenous fibrinolysis [93, 94]. In two different animal models of pulmonary thromboembolism in rabbits and mice we demonstrated that endogenous NO is an important regulator of agonist-stimulated platelet activation in vivo by reducing platelet activation and inducing platelet disaggregation in the pulmonary vasculature. In these models the effect of NO was mainly exerted at the level of platelets and not through an action on the vessel wall [95]. The importance of NO in preventing in vivo plateletmediated thrombosis was assessed by cross-transfusion



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experiments of platelets taken from wild type and knockouts for the eNOS gene that confirmed a role of platelet-derived NO in preventing platelet activation in vivo and in inhibiting the recruitment of new platelets to the growing thrombus [96]. Finally, it was shown that the antithrombotic action of NO in vivo is cGMP kinase-I mediated, in fact mice lacking the cGKI gene showed a much stronger tendency as compared to wild type animals to form arterial thrombi in response to ischemia/reperfusion [76].

oxidative stress; for most of these interventions an improving effect on endothelial NO biosynthesis was shown.

HUMAN STUDIES ON NO IN CARDIOVASCULAR DISEASE

a) Direct NO Donors

Conventional atherothrombotic risk factors, like smoking, aging, hypertension and dyslipidemia, are associated with impairment of platelet-derived NO release and a significant inverse correlation was found between platelet released NO and the number of risk factors present [97]. A reduction of NO release may in part be due to an oxidative stress which accompanies atherosclerosis [98] or to an altered intracellular redox state [99]. Vitamin C and reduced glutathione (GSH), potent antioxidants and oxygen free radical scavengers, modulate the action of NO; oxidative stress may be an important mechanism for impaired plateletderived NO bioactivity in chronic smokers [100]. Platelets of patients with acute coronary syndromes, such as myocardial infarction or unstable angina, produce significantly less NO than platelets of patients affected by stable coronary artery disease [87] suggesting that an impairment of NO production by platelets may contribute to acute coronary syndromes. Patients with symptomatic ischemic heart disease, and expecially acute coronary syndromes, exhibit increased platelet aggregability and decreased platelet responsiveness to the antiaggregatory effect of NO donors [101]. Elevated plasma homocysteine levels are associated with enhanced risk of atherothrombosis [102]. Elevated hyperhomocysteinemia has also been shown to suppress endothelial NO production [103] and platelet NO production [104]. Endothelial dysfunction has been proposed as a fundamental component of the pathophysiology of atherosclerosis (such as diabetes, hypercholesterolemia, hyperhomocysteinemia, hypertension, smoking) and it is mainly characterized by an impairment of NO synthesis or biologic activity. Endothelial dysfunction is associated with increased oxidative stress and to inflammatory chronic processes [105] and indeed NO reduces the endothelial expression of several inflammatory and adhesion molecules that increase plaque vulnerability [106] and play a role in the development of atherosclerosis [107]. The reduction of the anti-inflammatory potential of a damaged endothelium may contribute to plaque destabilization, a process that predisposes atherosclerotic plaques to rupture and that strictly depends on cellular plaque components and proinflammatory mediators [108]. The observation that several pharmacological interventions that improve endothelial function are associated with a decrease in cardiovascular events, independent of other risk factors modification, supports the concepts that cardiovascular risk factors share a common pathway that leads to endothelial dysfunction, such as

NO AS A THERAPEUTIC AGENT IN THE CARDIOVASCULAR SYSTEM The replacement of defective endogenous NO by NOdonating molecules or by compounds that stimulate NO biosynthesis represents one of the novel targets in cardiovascular pharmacologic research of the last few years.

Direct NO donors release spontaneously NO by their nitroso or nitrosyl functional group. This class of agents include NO gas, sodium nitroprusside (SNP) and sodium trioxodinitrate. NO gas is used by inhalation for pulmonary vascular disorders [109]; SNP contains the nitric oxide molecule in a nitrosyl group linked to iron and is used for the treatment of hypertension and heart failure [110]. The class of NONOate or diazeniumdiolate compounds contain NO covalently linked to diethylamine and diethylenetriamine [111]. Sydnonimines and furoxan compounds require cofactors to facilitate NO release, oxidants, such as molecular oxygen, and thiols, respectively [112]. S-nitrosothiols, including S-nitrosoglutathione, S-nitrosoN-acetylpenicilamine and S-nitroso-albumin, share many biological functions. They release nitric oxide, induce transnitrosilation and S-thiolation. Small clinical studies suggest that they can be of benefit in a variety of cardiovascular disorders [113]. b) Indirect NO Donors Indirect NO donors require metabolic activation to release NO. The classical nitrovasodilators used in the management of cardiovascular disorder include organic nitrite and nitrate esters like nitroglycerin, amyl nitrite, isosrbide dinitrite, isosorbide-5-mononitrate and nicorandil [114]. They induce vasorelaxation through their action on cGMP or by direct inhibition of non specific cation channels present in vascular smooth muscle cells. Most of these drugs require enzymatic metabolism to generate biological active NO. The enzymatic pathway involved in the denitration and reduction of organic nitrate esters to release NO is probably the cytocrome P450 system together with NADPH and glutathione-S-transferase [115, 116]. c) Compounds that Stimulate Endogenous NO Biosynthesis A number of drugs active in the prevention of ischemic cardiovascular events have been suggested to act in part also by an enhancement of endogenous NO production. Statins may improve endothelial function by lipid independent mechanisms, and recent reports suggest that this pleiotropic action of statins is operative in humans. Statins upregulate eNOS expression by different mechanisms: the activation of protein kinase B in endothelial cells which leads to phosphorylation of eNOS and to the subsequent increase of its activity [117] and the down regulation of caveolin-1

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expression, which acts as an inhibitor of eNOS activation by forming a heterocomplex with this enzyme [118]. Statins reduce the synthesis of farnesyl phyrophosphate and geranylgeranyl pyrophosphate [GGPP) by inhibiting Lmevalonate synthesis. GGPP is active in the posttranslational modification of a variety of proteins, including eNOS and Ras-like proteins, such as Rho, the inhibition of which results in a 3-fold increase in eNOS expression and nitryl generation [119]. Angiotensin–converting enzyme inhibitors (ACEIs) increase NO bioavailability by decreasing the synthesis of angiotensin II and by enhancing serum levels of NOreleasing bradikynin via inhibition of its degradation. Controversial results have been reported regarding the effect of angiotensin receptor antagonists, which specifically block the effect of angiotensin II on the angiotensin type I (AT I) receptor, on endothelial function [120]. Antioxidant may also enhance NO biosynthesis [121]. Given that increased oxidative stress plays a pivotal role in the pathogenesis of endothelial dysfunction [122], administration of antioxidants would be expected to be a reasonable strategy to treat this disorder. d) NO-ASA: A Brief Summary A novel class of antithrombotic agents has recently been produced by adding a nitric oxide functional group to the antiplatelet agent aspirin. In vitro nitroaspirin has been shown to exert a wider antiplatelet action as compared with aspirin, inhibiting platelet activation induced by a wide range of platelet agonists, suppressing also platelet adhesion to collagen under flow conditions as well as high-shear stress-induced platelet activation, an action that the parent compound, aspirin, does not exert [81]. These activities seem to be largely due to the effects of the NO moiety and the enhancement of intraplatelet cGMP that it produces and to a synergism between the antiplatelet action of NO and that of aspirin [81]. Interestingly, nitroaspirin exerts a number of additional activities on other cells, such as an antiproliferative action on smooth muscle cells [123], or an inhibitory effect on the expression of tissue factor by monocytes [124], which may be of relevance in cardiovascular pharmacology. Nitroaspirin has been tested in several animal models of thrombosis and/or atherogenesis, with interesting results. Namely, studies in the rat have shown antithrombotic activity in an extracorporeal circuit upon chronic administration of the drug [125]. We have shown that nitroaspirin is effective in preventing platelet pulmonary thromboembolism in vivo, in mice and rabbits [126]. We have compared in the same model the effect of aspirin, NCX 4016, and two direct NO donors, isosorbide-5-mononitrate (ISMN) and sodium nitroprusside (SNP) confirming that NO donating compounds inhibit the mortality induced by a mixture of collagen plus adrenaline and U46619, a stable analog of thromboxane A2, more than aspirin and that the protective effect of NCX 4016 is in part due to the vasoactive action of NO released from the molecule, an activity, however, that due to the slow pattern of NO-release does not induce any significant systemic vasodilation.

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Interestingly, the strong antithrombotic effects of nitroaspirin are not accompanied by a striking prohemorrhagic activity [127]. In addition, the combination of nitroaspirin with the ADP-receptor antagonist, platelet inhibitor clopidogrel, a thienopyridine, leads to a further enhancement of the antithrombotic activity which appears to be larger than that observed with the combination aspirin plus clopidogrel [127]. Nitroaspirin also exerts tissue protective effects in ischemia/reperfusion models, such as focal cerebral ischemia in the spontaneous hypertensive rats [128] or myocardial ischemia in the rabbit or rat [129] or even in the anesthetized pig [130]. Finally, and very interestingly, nitroaspirin has proven to be able to prevent restenosis and atherogenesis in hypercholesterolemic mice or aged rats [60, 61], in ApoE knockout mice [131] or even in normal mice after localized, oxygen radicals-induced arterial damage [132]. The strong antithrombotic effects of nitroaspirin are not accompanies by any gastric toxicity, which instead it typically associated with the use of aspirin, thanks to the mucosal protective effects of nitric oxide [133]. Finally, NO-aspirin has proven an effective antiplatelet agent in humans without simultaneously exerting the gastrotoxic properties of aspirin [134]. A phase I study in healthy volunteers has shown antiinflammatory effects, not shared by aspirin, on a number of cytokines and other inflammatory markers elicited by the i.v. administration of LPS [135] and now phase II studies in peripheral arterial disease, acute coronary syndromes and diabetes with this compound are ongoing. CONCLUSIONS Nitric Oxide a molecule discovered only 20 year ago, has acquired great importance, especially in the cardiovascular system where it has been demonstrated to regulate several physiologic phenomena and where a defect of its production or biologic activity contributes to important pathologic phenomena leading to ischemic cardiovascular disorders. The number of studies on Nitric Oxide is increasing in an exponential way and many important new discoveries in the basic biochemical reactions, physiologic role and pharmacologic applications are continuously accumulating. In particular, great effort has been put in the identification of therapeutic regimens able to release Nitric Oxide within the circulation and/or to facilitate the biosynthesis of endogenous NO. Many promising novel pharmacologic approaches are now available and large clinical trials proving the clinical advantage of these new treatments over available therapeutic regimens are now expected. REFERENCES [1] [2]

Suslick,K.S.; Reinert,T.J.The synthetic analogs of O2-binding heme proteins. J. Chem. Ed., 1985, 974. Spiro, T.G; Zgierski; M.Z.;Kozlowski,P.M.Stereoelectronic factors for NO from vibrational spectroscopy. Coord.Chem. Rev. 2001, 219, 923.

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