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Reactive Oxygen Species

来源:动脉硬化血栓血管生物学杂志 作者:Florian Kr?tz; Hae-Young Sohn; Ulrich Pohl 2007-5-18
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摘要: It is not surprising, therefore, that the platelet activation cascade (including receptor-mediated tethering to the endothelium, rolling, firm adhesion, aggregation, and thrombus formation) is tightly regulated。 In addition to already well-defined platelet regulatory factors, such as nitric oxid......


From the Institute of Physiology (F.K., U.P.) and Cardiology Division (F.K., H.-Y.S.), Medizinische Poliklinik-Innenstadt, Ludwig-Maximilians-University, Munich, Germany.

ABSTRACT

Platelets participate not only in thrombus formation but also in the regulation of vessel tone, the development of atherosclerosis, angiogenesis, and in neointima formation after vessel wall injury. It is not surprising, therefore, that the platelet activation cascade (including receptor-mediated tethering to the endothelium, rolling, firm adhesion, aggregation, and thrombus formation) is tightly regulated. In addition to already well-defined platelet regulatory factors, such as nitric oxide (NO), prostacyclin (PGI2), and adenosine, reactive oxygen species (ROS) participate in the regulation of platelet activation. Although exogenously derived ROS are known to affect the regulation of platelet activation, recent data suggest that the platelets themselves generate ROS. Intracellular ROS signaling in activated platelets could be of significant relevance after transient platelet contact with the vessel wall, during the recruitment of additional platelets, and in thrombus formation. This review discusses the potential cellular and enzymatic sources of ROS in platelets, their molecular mechanisms of action in platelet activation, and summarizes in vitro and in vivo evidence for their physiological and potential therapeutic relevance.

Platelets participate not only in thrombus formation but also in the regulation of vessel tone, the development of atherosclerosis, angiogenesis, and in neointima formation after vessel wall injury. It is not surprising, therefore, that the platelet activation cascade is tightly regulated. In addition to already well-defined platelet regulatory factors, such as nitric oxide (NO), prostacyclin (PGI2), and adenosine, reactive oxygen species (ROS) participate in the regulation of platelet activation. This review discusses the potential cellular and enzymatic sources of ROS in platelets, their molecular mechanisms of action in platelet activation, and summarizes in vitro and in vivo evidence for their physiological and potential therapeutic relevance.

Key Words: platelets ? reactive oxygen species ? NAD(P)H-oxidase ? aggregation ? adhesion ? thrombus formation

Introduction

Platelet interaction with the vessel wall serves numerous physiological and pathophysiological functions. This is reflected by the fact that platelets release growth factors,1 lipid mediators,2,3 and cytokines.4 Consequently, the regulation of platelet activity plays a role not only for thrombus formation and the regulation of vascular tone5 but also for the vascular pathophysiology of angiogenesis and inflammation. Moreover, platelets participate in the development of atherothrombotic disease by promoting atherosclerotic lesion6 and neointima formation.7 Not surprisingly, the activation of platelets is regulated and modulated by numerous factors, blood-borne and cell-derived. Most of these factors are relatively well-characterized.8 However, in recent time, several publications have suggested that reactive oxygen species (ROS) represent a new modulator of platelet activity. It has been known for some time that ROS exert critical regulatory functions within the vascular wall and it is therefore plausible that platelets represent a relevant target for their action.

Within the vessel wall (where endothelial cells, vascular smooth muscle cells, and fibroblasts express a variety of ROS-generating enzymes), there is a constant, low-quantity flux of ROS. It is already established that enhanced ROS release from the vascular wall can indirectly affect platelet activity by scavenging nitric oxide (NO), thereby decreasing the antiplatelet properties of the endothelium.9 In addition to their exposure to ROS derived from the vascular wall, platelets themselves also can generate ROS, and there is some evidence for a more direct role of ROS in the control of platelet activity. Finally, under inflammatory conditions, platelets are also exposed to phagocyte-dependent "burst-like" production of high quantities of ROS.10,11 With these circumstances in mind, it is surprising that our understanding of the influence of ROS on platelets lags far behind that of other soluble factors present in the vascular bed.

This review focuses on current knowledge regarding ROS-dependent regulation of platelet activity and their role in platelet adhesion and aggregation in vitro and in vivo.

ROS and Platelet Function

Influence of Exogenous ROS on Platelet Function

Although often referred to as a single entity, the term ROS actually comprises several factors that all potentially exert different vascular effects.12 The superoxide anion (O2–) is central to ROS chemistry, because it may be converted into other physiologically relevant ROS by enzymatic or nonenzymatic reactions. For example, O2– can react rapidly with NO to form peroxynitrite (ONOO–). It can also be converted to hydrogen peroxide (H2O2) by superoxide dismutase (SOD).12 Theoretically, both SOD and NO compete for O2– scavenging. Thus, it is worth mentioning that the rate of O2– conversion to H2O2 catalyzed by SOD is only one-third of the reaction rate of O2– with NO.13 H2O2 also serves as a substrate for the production of further detrimental ROS, such as hypochlorous acid (HOCl), generated by enzymatic conversion through neutrophil myeloperoxidase, and plasma levels of myeloperoxidase possess predictive value for coronary thrombotic events.14 Aside from enzymatic conversion, H2O2 reacts with ferrous iron (Fe2+), generating ferric iron (Fe3+) and the hydroxyl radical (OH–, "Fenton reaction").15 In the presence of catalase, H2O2 is degraded to water and oxygen. Aside from SOD or catalase, glutathione peroxidase (GPx) also exerts antioxidant enzymatic function, because it catalyzes a reaction that degrades H2O2 by oxidizing reduced glutathione (GSH) to its disulfide form (GSSG). Therefore, SOD, catalase, and GPx represent important antioxidant enzymes within vascular biology.

Several in vitro experimental approaches have been used to investigate the specific roles for distinct ROS on platelets. A simple approach to identify the role of H2O2 has been to directly expose platelets to H2O2, which, unlike O2–, ONOO–, or OH–, is relatively stable, diffuses through membranes, and is available as a chemical reagent. Divergent results have been obtained from experiments using exogenous H2O2. It inhibited ADP-dependent platelet activation,16,17 whereas it enhanced collagen-dependent or arachidonic acid (AA)-dependent platelet activation.18 This discrepancy may be caused by the different antioxidant capacities of the buffers used.

In addition to direct administration of H2O2, platelets have been exposed to O2–-generating enzymatic systems such as glucose/glucose oxidase, (hypo-)xanthine/xanthine oxidase (XO), or pyrogallol. Thereby, several independent studies have shown that O2– reduces the threshold for platelet activation to thrombin, collagen, ADP, or AA, and may even induce spontaneous aggregation.19–22 In all cases, it appeared that O2–, but not OH– or H2O2, mediated the enhanced platelet activity.21,22 In a more recent study, Pratico et al23 demonstrated that an alternative ROS, OH–, was generated during full blood platelet aggregation induced by collagen, and that the OH– scavenger mannitol partly prevented this aggregation, suggesting that >1 ROS may be involved in enhancing collagen-dependent platelet activation.

In addition to directly activating platelets or decreasing the threshold for platelet activation, O2– reacts with platelet or endothelium-derived NO to ONOO–, which is of particular importance for vascular thrombosis. This is primarily caused by the decreased bioavailability of NO as a potent inhibitor of platelet activation. It is not surprising, therefore, that the antithrombotic effect of NO is lost either when ROS are exogenously added to the system or when they are scavenged by SOD.24,25 It is, however, likely that ONOO–, being a highly reactive molecule, exerts additional effects on platelets. Direct administration of peroxynitrite to platelets inhibits the aggregation of platelets induced by ADP, thrombin, collagen, and other platelet stimuli.26,27 However, ONOO– appears to possess a dual effect: whereas it activates platelets in normal buffer, this effect is reversed when plasma is added.27,28 In some cases, inhibitory effects of ONOO– are likely caused by the antiplatelet activity of S-nitrosothiols,29 which are formed when ONOO–-generating drugs like SIN-1 are applied.30 Unfortunately, a specific effect of ONOO– generated during the reaction of endothelial NO with O2– has not been investigated in mixed suspensions of these cells in vitro yet.

Notably, results from in vitro exposure of platelets to exogenously applied H2O2, O2–, or ONOO– must be interpreted cautiously. First, the use of high, presumably nonphysiological concentrations of H2O2 or other ROS induces leakage of intracellular contents via membrane disruption (such effects may occur at H2O2 concentrations of 0.1 mmol/L and higher).31 Some reported inhibitory effects of H2O2 on platelet aggregation may have been caused by this effect.32,33 Second, the antioxidant capacity of the assay buffer must be considered when working with oxidants and platelets. Although studies with washed platelets allow for "unaltered" information about the function of a specific ROS, this scenario is not necessarily representative of physiological conditions, because plasma and full blood possess antioxidant capacities.34,35 However, studies of full blood may be biased by oxidants and antioxidants derived from cellular sources other than those from platelets. Finally, some data obtained in the presence of SOD, which is normally applied to identify the specific action of O2–, may also be explained by the effects of an increased concentration of H2O2, which is generated by the reaction of SOD with O2–. Thus, awareness of the assay conditions is of importance for the interpretation of data regarding the influence of ROS on platelets. If the influence of a specific ROS on platelets is investigated in vitro, then buffers that do not contain antioxidant substances may be appropriate. If consequences of such signaling should be assessed with relevance for the in vivo situation, however, then the effects of addition of defined amounts of plasma or full blood should be tested.

Role of Platelet-Derived ROS

In addition to ROS derived from exogenous sources, ROS are also generated by activated platelets. The release of O2– and other ROS by platelets was first observed by Marcus in 1977.36 In the following years, the release of several ROS, including O2–, OH–, and H2O2 from platelets was reported, both from unstimulated platelets and after platelet stimulation with agonists such as collagen or thrombin.37–39 This endogenous formation of ROS suggests they have autocrine or paracrine roles in platelet activation similar as described for exogenous ROS.

Like endothelium-derived ROS, platelet-derived ROS potentially stem from different enzymatic sources (summarized in Figure 1), but one of them, the platelet isoform of NAD(P)H-oxidase, has gained most attention, because it can be activated on stimulation by agonists that also induce platelet activation.20,31,40–42 Evidence for the expression of an NAD(P)H-oxidase enzyme in platelets first arose from the observation that diphenylene iodonium chloride, a rather nonspecific inhibitor of flavoprotein-dependent enzymes (among them NAD(P)H-oxidase), inhibited platelet aggregation.43 Inhibition of platelet O2– formation by diphenylene iodonium chloride has been shown independently by several groups.44,45 The first direct evidence for the expression of a platelet NAD(P)H-oxidase was presented by Seno et al, who detected p22phox and p67phox, 2 subunits of the NAD(P)H-oxidase enzyme (there are at least 5 known subunits), in platelet lysates.46 The expression of a platelet isoform of NAD(P)H-oxidase was further underpinned by detection of the gp91phox and p47phox subunits.20,47 The specific inhibition of the gp91phox subunit could block platelet O2– production in a study that also demonstrated that collagen stimulation indeed increased platelet NAD(P)H-oxidase activity.20 Furthermore, NAD(P)H-oxidase–dependent platelet O2– production enhanced the recruitment of platelets to a growing thrombus, most likely by inactivating a platelet ectonucleotidase, thereby increasing bioavailability of ADP.20 Notably, the magnitude of NAD(P)H-oxidase derived O2– flux is in the nanomolar range,20 and thus it is similar to the flux that is present in endothelial cells48 but <1% of the amount of O2– released from activated neutrophils.49 When total release is measured in endothelial cell monolayers or defined platelet suspensions, again, levels of platelet O2– are similar to those produced by endothelial cells.20,48

Figure 1. Enzymatic sources for reactive oxygen species (ROS) in the vascular endothelium and in platelets. Several enzymatic systems contribute to the production of ROS, thereby influencing platelet activity. In the endothelium (EC), NAD(P)H-oxidase, cyclooxygenase isoforms 1 and 2 (Cox), cytochrome P450 epoxygenase isoform 2C9 (CYP2C9), xanthine oxidase (XO), uncoupled endothelial NO synthase (unc. eNOS), and mitochondrial respiration contribute to the production of superoxide radicals (O2–), hydrogen peroxide (H2O2), and hydroxyl radicals (OH–). Platelets also contain NAD(P)H-oxidase activity, and many of its subunits have been found in platelets at the protein level (Nox2 or gp91phox, p22phox, p67phox, p47phox, and Rac). Platelet NAD(P)H-oxidase is stimulated on collagen (most likely mediated by GPVI) or thrombin exposure (most likely mediated by PAR1) and after membrane depolarization. It is unclear which of the Rac isoforms participates in platelet NAD(P)H-oxidase activation. Other potential sources for ROS from platelets are cyclooxygenase-1, xanthine oxidase, mitochondrial respiration, or uncoupled eNOS (when there is insufficient supply of cofactors such as tetrahydrobiopterin ). In addition, recent data indicate that there is redox potential-dependent regulation (GSH/GSSG ratio) of the platelet fibrinogen receptor (GPIIb/IIIa). Transparent symbols indicate that these enzymes have not been detected on a protein level to date, although there is pharmacological evidence for their existence. Note that macrophages, which are not shown in this Figure, are another important source for ROS with relevance to platelets.

Recently, Clutton et al presented evidence that on stimulation with a thrombin-receptor activating peptide platelet phosphatidylinositol 3 kinase (PI3-kinase)-induced membrane translocation of platelet p67phox and thus regulated NAD(P)H-oxidase–dependent O2– release from platelets.50 Inhibition of phosphatidylinositol 3 kinase41,50 or protein kinase C20,39,41 have been found to decrease O2– release from platelets, whereas direct activation of protein kinase C resulted in enhanced platelet O2– production.20,39,41 Membrane depolarization also stimulated O2– production in platelets,51 similar as observed in endothelial cells, in which depolarization stimulated O2– production was found to be caused by a rac1-dependent activation of NAD(P)H-oxidase.48,52,53 Accordingly, we were able to show that platelet hyperpolarization prevented platelet-derived ROS release and platelet adhesion to endothelial cells in vitro.54

Aside from NAD(P)H-oxidase, a variety of other enzymes is capable of producing ROS in platelets, as in most other cells of the vasculature. The endothelial isoform of NO synthase (eNOS) is a well-characterized source of O2– in endothelial cells when there is a shortage of cofactor supply ("uncoupling"),12 and there is evidence that eNOS may also be a source of platelet-derived O2– because platelets from eNOS-deficient mice show a markedly reduced O2– release.55 Furthermore, it has been shown that XO was not only present in platelets but also contributed to thrombin-induced platelet ROS production.39 Other observations imply an additional role for phospholipase A2 (PLA2)-dependent AA release contributing to ROS production.37 By analogy, this may be explained by the fact that the AA-converting prostaglandin H-synthase that also exists in platelets contains a cyclooxygenase and peroxidase moiety within its enzymatic complex,56 which can generate significant amounts of O2–.57 It is unknown, however, whether this really occurs in platelets. Other enzymes of AA metabolism, such as lipoxygenases, may also participate in platelet ROS release,58,59 but again, there is no evidence so far for a functional role of lipoxygenase or cyclooxygenase-dependent platelet ROS formation. Thus, in some cases, it remains to be shown that the release of ROS is not merely an epiphenomenon that does not necessarily play a functional role.

Role of ROS in Platelet Signaling

The hypothesis of an important role of platelet-derived ROS in platelet activation was initially supported by studies applying antioxidant enzymes to inhibit platelet activity. SOD, an O2–-degrading enzyme, has occasionally been observed to have an inhibitory effect on agonist-induced platelet activity when applied exogenously to platelet suspensions; however, the effect of SOD on platelets differed depending on the stimulus used.20,60

Some of the conflicting data regarding the influence of platelet derived O2– on platelet aggregation can be explained by the kinetics of platelet ROS production after stimulus exposure. Platelet O2– production on collagen stimulation occurs with a delay of 3 to 5 minutes,20 so the involvement of platelet O2– in the initial steps of aggregation may be minimal. In the case of collagen, the aggregation starts only after a 1- to 2-minute lag phase after stimulus exposure. Although there was no effect of platelet-derived O2– in this initial phase of aggregation, late thrombus growth was significantly increased because O2– increased the bioavailability of ADP, leading to an increased recruitment of additional platelets.20 Similar results were reported after stimulating platelets with a thrombin receptor-activating peptide: the concomitant O2– release from platelets prevented late thrombus disaggregation that is normally caused by platelet-derived NO.50 There is accumulating evidence that platelet-derived O2– is a functionally relevant scavenger of platelet-derived NO, which inhibits the anti-aggregatory effects of platelet-derived NO.45,61 In accordance with this, GPIIb/IIIa inhibitors decrease platelet O2– release and enhance platelet NO release concomitantly.61 Some of these observations also indicate that an effect of platelet-derived ROS on platelet aggregation may play a prominent role in a late phase of aggregation. Although the mechanism by which O2– induces recruitment and enhanced platelet aggregation is not yet fully elucidated, it is likely that it involves scavenging of platelet NO by O2–,50 and/ or regulation of redox-sensitive ectonucleotidases located on platelet and endothelial cell membranes20,48,52,53 (Figure 2).

Figure 2. Recruitment of platelets to a growing thrombus is enhanced by superoxide. When coming into close contact with subcellular matrix components (black curved lines), such as collagen, resting platelets (ovals) tether to the endothelium and are activated. Activated platelets (bizarre shapes) release substances, such as ADP (or thromboxane A2), that lead to recruitment of additional circulating resting platelets (ovals). On stimulation, platelets also release ROS such as superoxide (O2–), which increase thrombus formation either by preventing the degradation of ADP (thereby also lowering the bioavailability of the platelet inhibiting ultimate degradation product of ADP, adenosine) or by degrading bioavailability of nitric oxide (NO). NO, under physiological conditions, prevents platelet activation by increasing intraplatelet cGMP levels. Platelet release of O2– can be prevented by GPIIb/IIIa inhibitors.

In studies using full blood, platelet activation on subthreshold exposure to collagen or AA was primed to result in irreversible aggregation when neutrophils were stimulated with N-formyl-Met-Leu-Phe.10 This effect could be prevented by the blockade of neutrophil NAD(P)H-oxidase and by catalase, supporting an additional role for H2O2.10,62 Of note, during the catalase reaction with H2O2, relevant amounts of the highly oxidized catalase intermediate "compound I" may be generated,63 which (similar to NO) is able to stimulate soluble guanylate cyclase.17,64 Such observations underpin the difficulties in identifying the specific effects of distinct ROS by using antioxidant enzymes.

The fact that platelets express antioxidant enzymes65 also suggests a role of ROS in platelet signaling, because these antioxidant enzymes are likely not only to prevent cytotoxic effects of ROS but also to regulate oxidation sensitive signaling pathways in platelets. One of these enzymes, GPx, has gained notable attention with respect to arterial thrombus formation, because an innate deficiency of plasmatic GPx has been observed in 2 brothers with childhood cerebrovascular thrombosis.66 In addition to its antioxidant properties, GPx also enhances the bioavailability of NO by catalyzing its liberation from s-nitrosothiols and reducing lipid peroxides.67 By using GSH as a cosubstrate, GPx not only reduces H2O2 to water but also reduces lipid peroxides to their respective alcohols.68 Although it is not clear whether this reaction occurs in living cells, O2– can directly react with GSH, resulting in a chain reaction that ultimately produces oxidized glutathione (GSSG) and additional O2–.69,70 Thus, O2– has the potential to even directly decrease the equilibrium between reduced and oxidized glutathione (GSH/GSSG ratio), being of high importance for the redox regulation of protein thiol groups. The majority of these thiols exist in a reduced form when the GSH/GSSG ratio is high, which is the typical intracellular scenario, where the ratio amounts to 100/1.34 Shifting the intracellular redox balance to a lower GSH/GSSG ratio increases platelet sensitivity to activating agents, as demonstrated by increased intracellular calcium spiking.71 Consequently, platelet supplementation with GSH decreases platelet aggregation in vitro.72–74 Further, N-acetyl-L-cysteine, which is capable of restoring intracellular thiol stores by shifting the redox balance in favor of GSH, inhibits platelet aggregation and potentiates the antiplatelet effects of NO,75 underscoring the critical role for the platelets’ intracellular redox state.

It is unclear at the moment which and how many proteins of importance for platelet function are potentially regulated by such redox-dependent mechanisms. There is, however, intriguing evidence that the IIb?3 integrin—a large transmembraneous heterodimeric glycoprotein receptor with a prominent role in platelet aggregation—is regulated by oxidants on several sites. The extracellular domains of IIb?3 are rich in disulfide bonds, which, on reduction, activate the receptor and induce a pro-aggregatory state, probably by stabilizing ligand–integrin interactions.76–78 The number of free thiols within IIb?3 is regulated by the extracellular GSH/GSSG ratio. Intriguingly, when the ratio is close to that in full blood (5/1), there is a maximum effect of agonist-induced aggregation.34 Recently, it has been described that platelets contain a glutathione reductase-like activity on their surface, which participates in regulating the extracellular GSH/GSSG ratio, and is capable of generating the appropriate redox potential for platelet activation.79 Similar mechanisms have been observed to regulate collagen-dependent platelet adhesion to the 2?1 integrin80 and are proposed to participate in inactivation of the platelet P2Y12 receptor.81

Independent from this, another oxidative platelet activatory mechanism involves the short intracellular tail of ?3, which can be tyrosine phosphorylated on extracellular ligand binding.82 This has been suggested to represent an important step in platelet outside-in signaling, as it was associated with enhanced binding of tyrosine kinase adapter proteins such as GRB2.82 This tyrosine phosphorylation of ?3 could also be induced by H2O2 (in combination with a tyrosine phosphatase inhibitor), which resulted in spontaneous platelet aggregation.83

Lastly, ex vivo studies indicate that antioxidant vitamins exert inhibitory actions on platelet function.84 Vitamin E (-tocopherol) inhibits platelet aggregation and the release reaction,85 suggesting that these effects were caused by a decreased O2– bioavailability in platelets.55 Vitamin C (ascorbic acid) and the red-wine phenolic antioxidant transresveratrol reportedly also reduced production of O2– in platelets.86 Recent reports also indicate that oral vitamin C administration can inhibit platelet aggregation ex vivo,87 and transresveratrol slightly reduces platelet aggregation and platelet lipid peroxidation.88 However, vitamins E and C can also exert pro-oxidant effects,86,89 and it remains unclear whether the administration of "potentially antioxidant" vitamins always exerts antithrombotic effects in vivo. Randomized clinical trials investigating a protective role for these compounds in cardiovascular disease have been disappointing thus far.90,91

Thus, although redox regulation of platelet signaling is likely, a detailed understanding of redox-dependent platelet activatory signaling is at present limited to the data mentioned here.

Role of ROS in Platelet Activation In Vivo

Because of the concurrent presence of several distinct ROS, the actual influence of a specific ROS on platelet function in vivo may not necessarily match in vitro findings. Several lines of evidence, however, support the hypothesis that ROS are crucially involved in the physiology and pathophysiology of atherothrombosis in vivo.

It is clearly evident that various risk factors for atherosclerosis and cardiovascular thrombosis, such as hypertension, hypercholesterinemia, hyperglycemia, cigarette smoking, or hyperhomocysteinemia, are associated with ROS-mediated development of endothelial dysfunction92,93 (a list is given in the Table). Because of their mass, platelets tend to flow outside the central high-velocity axis of laminar blood stream,94 so a large number of platelet interactions with the endothelium occur spontaneously, even under nonthrombotic conditions.95 Endothelial dysfunction or damage by oxidants96 are then associated with an enhanced risk for platelet activation and subsequent atherothrombotic complications.9,97 In addition, in vitro and in vivo evidence suggests that ROS, apart from directly activating platelets, increase their adhesion to the vascular endothelium by degrading the endothelial glycocalix,98 by acting on transcriptional mechanisms in the endothelium,13,99 by inactivating endothelial ectonucleotidases (enhancing the bioavailability of ADP52), or by an ischemia reperfusion-dependent endothelial cell activation.99

Cardiovascular Risk Factors and Diseases Associated With Enhanced Platelet ROS Generation in Humans

A direct involvement of ROS in thrombus formation in vivo has also been reported. It was first observed by Yao et al, who showed that recombinant CuZnSOD, recombinant MnSOD, and catalase infusion decreased cyclic flow variations (CFV), a correlate for in vivo platelet activation, in the coronary arteries of dogs.60 Corroborating an influence of ROS on platelet function in vivo, platelet recruitment to a growing thrombus was decreased after intravenous administration of SOD and catalase in rats, whereas it was enhanced by inhibition of NOS.100 Yao et al also showed that catalase and SOD prevented platelet-dependent CFV, suggesting that both O2– and H2O2 enhance thrombus formation in vivo. Strikingly, in animals in which catalase had no effect, SOD worsened CFV, underscoring a central role of high concentrations of H2O2.60 These results were later confirmed by Ikeda et al.101 In both studies, radical fluxes were generated by xanthine/XO-induced CFV; it was also observed that an inhibitor of endogenous XO, allopurinol, was able to prevent CFV induced by ADP.102 It was determined that O2– derived from uncoupled platelet eNOS was involved in platelet activation during CFV.103 Evidence for a significant role for other platelet sources of ROS, such as NAD(P)H-oxidase, in vivo is still limited: recently, hypercholesterinemic mice deficient of gp91phox, which is the large membrane-bound electron transferring complex of NAD(P)H-oxidase, were observed to have reduced P-selectin–dependent platelet rolling, compared with hypercholesterinemic controls.104 Whether an increase of platelet rolling was caused by endothelial, neutrophil, or platelet NAD(P)H-oxidase activity remained unclear in these experiments.

Unfortunately, there are very few studies so far that give direct clues for the mechanisms underlying the prothrombotic effects of ROS in vivo. Several studies reported enhanced lipid peroxidation in association with platelet O2– formation and activation,42,105–108 but this is only a rather unspecific marker, similar as the reported decrease of plasmatic thiol levels associated with platelet O2– formation.109–111 Notably, plasma redox state is influenced by several plasmatic thiols such as homocysteine or cysteine; therefore, it is difficult to correlate the source of altered plasmatic redox state to thrombotic risk.

Conclusions

Overall, there is accumulating evidence supporting a net prothrombotic effect of vascular-derived and platelet-derived ROS in vitro. Several lines of evidence also suggest an oxidative enhancement of thrombus formation in vivo. Taken together, these data point to an important role of platelet-derived ROS and the intraplatelet redox state in the regulation of physiological platelet activation. In several cardiovascular disease states, augmented interactions of oxidants with platelets and the release of ROS from platelets have been observed, further supporting a causal role of enhanced ROS production in platelet pathophysiology.

However, because of the complexity of ROS chemistry and because of the great number of regulatory enzymatic sources that are potentially involved, many questions about the role of ROS for platelet activation and the role of the different ROS-producing enzymatic systems remain unclear. In particular, the specific significance of the different types of ROS with respect to platelet activity has not definitely been determined. This is especially true for H2O2 and ONOO–, whereas the data concerning a stimulatory role for O2– or OH– are more consistent. The actual influence of ROS on biochemical redox levels also needs further attention because the GSH/GSSG ratio currently emerges as one potentially important regulator of platelet signaling. In addition, the specific role of oxidant-dependent platelet regulation and oxidant producing enzymes within platelets in comparison to other platelet regulatory mechanisms has not been evaluated. In some situations, it thus remains to be confirmed that platelet ROS production actually exerts a specific pathophysiological function, rather than simply existing as an epiphenomenon. However, because O2– fluxes from platelets are similar to those observed in endothelial cells, and because both cell types exhibit some similarity in terms of equipment with oxidant enzymes, it seems likely that the significance of platelet O2– is underestimated so far. This is even more so, because the role of platelets for atherogenic, inflammatory, or angiogenic processes, which are all likely to be influenced by platelet-derived ROS, is only gradually being defined.6,7,112–115 It will also be of significant interest to determine whether therapeutic intervention to reduce platelet ROS formation will be of use for antithrombotic treatment strategies, as suggested recently.61 Platelet ROS formation then could potentially be of use as a predictor for the progression of atherothrombotic disease.

Although it is clearly evident that ROS participate in platelet activation and subsequent thrombus formation, it is not fully understood whether ROS serve as indispensable signaling molecules for specific platelet activation pathways. Possibly, the mere presence of ROS in the vicinity of platelets defines the conditions of the "physiological battleground" on which platelets encounter their physiological/pathophysiological destinations.

Acknowledgments

The authors thank Dr Darcy Lidington for critically reading this manuscript and for giving important suggestions for improvement.

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