Skip Navigation

Cardiovascular Research 1997 34(3):445-452; doi:10.1016/S0008-6363(97)00101-6
© 1997 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Ambrosio, G.
Right arrow Articles by Golino, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ambrosio, G.
Right arrow Articles by Golino, P.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 1997, European Society of Cardiology

Reactive oxygen metabolites and arterial thrombosis

Giuseppe Ambrosioa,*, Isabella Trittoa and Paolo Golinob

aDivision of Cardiology, University of Perugia School of Medicine, Perugia, Italy
bFederico II School of Medicine, Naples, Italy

* Corresponding author. Sezione di Cardiologia ‘R’, Dipartimento di Medicina Clinica, Via Eugubina 42, 06122 Perugia, Italy. Tel.: +39-75-5855842; fax: +39-75-5855840; e-mail: cardiopg@unipg.it

Received 14 October 1996; accepted 27 March 1997


   Abstract
Top
 Abstract
1 Characteristics of reactive...
 2 Effects of oxidants...
 3 Effects of oxidants...
 4 Effects of oxidants...
 5 Effects of oxidants...
 6 Leukocytes and thrombus...
 7 Conclusions
 References
 
Arterial thrombus formation is the result of complex events which require the interaction of damaged vessel walls with blood cellular elements and coagulation factors, and in which several mediators may play a role. In this context, the role of ‘classical’ chemical mediators such as thrombin, thromboxane or serotonin in initiating and/or amplifying intravascular thrombus formation is well established. However, it is now being recognized that certain chemical species formed in the metabolism of oxygen may also be involved in the process of arterial thrombosis. This review will focus on recent evidence in this field.


   1 Characteristics of reactive oxygen metabolites
Top
 Abstract
 1 Characteristics of reactive...
2 Effects of oxidants...
 3 Effects of oxidants...
 4 Effects of oxidants...
 5 Effects of oxidants...
 6 Leukocytes and thrombus...
 7 Conclusions
 References
 
Aside from H2O and CO2, which can be considered chemically ‘inert’, metabolism of O2 can give rise to various species which instead are extremely reactive [1]. One major category is represented by oxygen radicals, which are chemical species characterized by the presence of an unpaired electron. Oxygen radicals can attack and oxidize most cell components. This, in turn, may have profound effects on cell structure and function. Small amounts of superoxide radicals (•O2–) are continuously formed in vivo, a major source being mitochondrial respiration [1, 2]. Generation of superoxide has also been associated with certain steps in prostaglandin synthesis, particularly with the activity of prostaglandin hydroperoxidase [3]. Once formed, superoxide radicals can give rise to hydrogen peroxide (H2O2), a non-radical yet oxidant species, and to the powerful oxidant hydroxyl radical (•OH). Leukocytes are another major source of oxidants, because upon activation they generate H2O2, •O2–, and hypochlorous acid [4].

In addition to being produced in small amounts under physiologic conditions, large amounts of reactive oxygen metabolites can be released in the vascular lumen following restoration of flow after a period of ischemia [5, 6]. It has been proposed that this oxidant load might be responsible for a specific form of reperfusion-mediated tissue injury, secondary to lipid peroxidation and other irreversible alterations of cell constituents [7–9]. However, it is now appreciated that, apart from inducing gross cell damage, oxidants might also exert other effects which may be mediated through activation or inhibition of various enzymes [10]. Some of these effects may play a role in intravascular thrombosis, and oxidants may influence the process of thrombus formation by interfering at multiple steps with its various components (i.e., platelets, vessel wall, and coagulation factors), as discussed below.


   2 Effects of oxidants on platelet function
Top
 Abstract
 1 Characteristics of reactive...
 2 Effects of oxidants...
3 Effects of oxidants...
 4 Effects of oxidants...
 5 Effects of oxidants...
 6 Leukocytes and thrombus...
 7 Conclusions
 References
 
Platelets, a major player in thrombus formation, are obviously a prime target for oxidants produced or released in the vascular lumen and, at the same time, they are also capable of endogenous generation of oxidants [11, 12]. Thus, it is important to characterize the effects of oxidants on platelet function. Earlier studies investigating the effects of oxidants on platelet aggregation in vitro reached conflicting conclusions. While several investigators described an inhibitory effect of oxidants on platelets, other reports suggest that reactive oxygen species might actually enhance aggregation. Which oxidant species was responsible for the reported effects was also debated. Much of the controversy is probably related to differences in the experimental conditions employed in those studies, such as concentration and chemical nature of oxidants tested, or the effects of plasma components. Furthermore, it is now clear that oxidants can affect several key steps of platelet function, and therefore their net effect on the final process of aggregation may depend on various factors.

2.1 Role of different oxidant species
As already mentioned, earlier studies have investigated the effects of reactive oxygen metabolites on platelet aggregation. Several investigators observed inhibition of platelet function upon exposure to H2O2 [13–15], while others suggested that it might actually enhance aggregation [12, 16, 17]. Furthermore, studies can also be found that indicate that exposure to H2O2 has no effects on platelets, and that instead superoxide radicals may enhance aggregation [18–20]. Much of the controversy seems attributable to important differences in the experimental protocol, as discussed below.

In the attempt to clarify these discrepancies, we have recently conducted experiments aimed at systematically assessing the effects of different oxidant species on platelet function under pathophysiologically relevant conditions [21]. To this goal, we studied platelets in the presence of plasma, since this experimental model more closely resembles the physiological condition. In this system we investigated separately the effects of either superoxide radicals or H2O2, at biologically relevant concentrations. Platelet function was studied after a 60 s exposure to exogenously generated oxidants at a flux of 15–30 nmol/min. This protocol was designed to reproduce the duration and magnitude of oxidative stress as it may occur in vivo [5, 22]. It was found that H2O2, but not superoxide radical, can profoundly yet reversibly impair platelet aggregation in response to various agonists [21] (Fig. 1). With respect to previous studies, our data confirm that H2O2 has an inhibitory role when platelets are exposed to concentrations of this oxidant comparable to those achieved during oxidative stress [13–15]. As for the lack of effect of superoxide radical, it should be pointed out that studies showing a pro-aggregatory role of this species typically used washed (or gel-filtered) platelets resuspended in buffer [18–20], that is a system lacking the endogenous scavengers which are physiologically present in plasma. Our data [21] and experiments by Salvemini et al. [20] demonstrate instead that the aggregatory action of superoxide radical is lost when platelets are resuspended in plasma, thus indicating that the effects of superoxide radical on platelets may have limited pathophysiological relevance.


Figure 1
View larger version (33K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Effects of incubation with exogenous reactive oxygen metabolites on platelet aggregation. Values are mean±s.e.m. of 8–12 experiments. Platelet aggregation is expressed as percent of maximal aggregation under control conditions, and after 60 s of incubation with the oxidant-generating system xanthine/xanthine oxidase, with or without scavengers. Data are from [21]. Top panel: Platelet aggregation in response to ADP. Middle panel: Platelet aggregation in response to collagen. Bottom panel: Platelet aggregation in response to the thromboxane mimetic U46619. Under all conditions, exposure to oxidants significantly impaired aggregation. This effect was prevented by the H2O2 scavenger catalase, but not by superoxide dismutase. X/XO=xanthine 400 µM+xanthine oxidase 100 mU/ml; SOD=superoxide dismutase 1000 U/ml; CAT=catalase 2000 U/ml. *P<0.05 vs. controls.

 
2.2 Role of cyclic nucleotides
A major mechanism controlling platelet aggregation is represented by the activity of adenylate cyclase and of guanylate cyclase. Exposure to oxidants of plasma-resuspended platelets had no effects on basal cAMP levels [21]. Similarly, cAMP concentration showed the expected decrease following stimulation [21]. In contrast, impaired aggregation by H2O2 was accompanied by a >10-fold increase in platelet concentration of cGMP [21], consistent with the notion that H2O2 can stimulate guanylate cyclase in other cell types [23] and that stimulation of cGMP production can exert inhibitory effects on platelets in response to various agents, such as prostacyclin and nitrates [20, 24, 25]. That stimulation of guanylate cyclase was responsible for the inhibitory effect of H2O2 seen in our study is further supported by the observation that exposure to H2O2 of platelets that had been preincubated with the inhibitor of guanylate cyclase LY-83583 was associated with normal aggregatory response [21].

2.3 Role of cyclooxygenase
The activity of cyclooxygenase (the key enzyme of prostanoid metabolism) can be importantly affected by oxidants. This enzyme has the peculiarity of catalyzing two different reactions: oxygenation of arachidonic acid to PGG2, and the subsequent conversion of PGG2 to PGH2. Whereas the first reaction requires peroxides, PGH2 synthase activity is accompanied by formation of reactive oxygen species, which in turn may irreversibly inactivate the enzyme [3, 26]. We have also investigated the effects of oxidants on platelet cyclooxygenase activity.

Under our experimental conditions, superoxide radicals had no effects on platelet synthesis of thromboxane. In contrast, H2O2 played an important role. In fact, arachidonic acid-dependent aggregation was almost completely suppressed in the presence of catalase (a scavenger of H2O2) [21]. Furthermore, exogenously administered hydrogen peroxide markedly shortened the lag phase that precedes aggregation by arachidonic acid [21]. This lag phase corresponds to the lag phase between addition of arachidonate and synthesis of prostanoids demonstrated in partially purified preparation of cyclooxygenase, which can similarly be abolished by catalase or enhanced by peroxides [3, 26]. Low-level production of hydrogen peroxide seems therefore necessary to promote thromboxane synthesis, and hence platelet aggregation, in response to arachidonic acid stimulation. This conclusion is also supported by data by Del Principe et al. [12, 16] and by Praticò et al. [17], who showed that low concentrations of hydrogen peroxide could potentiate the aggregatory response to arachidonic acid or collagen.

In summary, the effects of reactive oxygen metabolites on in vitro platelet function are complex. Low levels of H2O2 may promote thromboxane synthesis and aggregation, whereas exposure to larger (albeit not toxic) concentrations of exogenous H2O2 may inhibit aggregation to several agonists via stimulation of guanylate cyclase and increased cyclic GMP formation.

2.4 Role of other platelet agonists
Another possible mechanism by which oxidants may influence platelet aggregation is through potentiation of the effects of platelet-activating factor (PAF). PAF is an autacoid released by platelets and other cell types (e.g., endothelium, leukocytes), which acts on platelets at extremely low concentrations (10–11–10–10 M). We have recently shown that superoxide and hydroxyl radicals can rapidly (i.e., within seconds) and irreversibly inactivate plasma PAF-acetylhydrolase, the enzyme that catabolizes PAF [27] (Fig. 2). Once PAF is formed or released in the blood, inhibition of PAF-acetylhydrolase would enhance concentrations and prolong half-life of this powerful agonist. Consistent with this hypothesis is the observation that neutrophil recruitment and activation induced by PAF in the mesenteric circulation is substantially reduced by administration of scavengers [28]. Thus, it may be speculated that oxygen radicals may indirectly enhance platelet aggregation, through local increases in PAF concentrations secondary to reduced breakdown of PAF. In vivo this hypothesis is indirectly supported by data derived from a dog study reviewed below [29], in which administration of superoxide dismutase was associated with a significant reduction in PAF-mediated aggregation of platelets resuspended in plasma, consistent with preserved activity of plasma acetylhydrolase.


Figure 2
View larger version (37K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Effects of exposure to oxidants on PAF-acetylhydrolase activity. Enzyme activity (measured from conversion of [3H]PAF to [3H]lyso-PAF) was virtually abolished by incubation with xanthine (X; 200 µM) plus xanthine oxidase (XO; 100 mU/ml). In parallel experiments, oxidants were inactivated by the specific scavengers superoxide dismutase (SOD; 330 U/ml), catalase (CAT; 1000 U/ml), dimethylthiourea (DMTU; 10 mM), or the iron chelator deferoxamine (DFX; 100 µM). Efficacy of SOD, DMTU, and deferoxamine indicates that PAF-acetylhydrolase is inactivated by hydroxyl radicals formed in a superoxide-driven, iron-catalyzed reaction. Data are from [27]. *P<0.05 vs. controls.

 
2.5 Comparison of in vitro vs. in vivo effects of oxidants on platelets
The complexity of the effects of reactive oxygen metabolites on platelets observed in vitro is reflected also in experiments conducted in vivo. The role of endogenous oxidants on intravascular thrombus formation has recently been examined in a canine model of coronary artery stenosis and endothelial injury [29, 30]. In this model, which mimics the situation potentially encountered in patients with unstable angina, episodes of gradual decrease in flow develop cyclically as a consequence of recurrent platelet aggregation/thrombus formation at the site of stenosis. Administration of the scavengers superoxide dismutase and catalase abolished these cyclic flow reductions in the majority of the animals. Compared with our in vitro data, these observations would confirm that under certain circumstances low concentrations of endogenously produced oxidants are required to promote platelet aggregation. However, it should also be noted that in one study administration of superoxide dismutase to those animals in which catalase failed, worsened cyclic flow reductions, indicating a more complex role of oxidants in intracoronary thrombus formation [29].

The role of exposure to exogenously administered oxidants on in vivo platelet aggregation has also been recently evaluated. In the same study by Willerson and associates [29], when a coronary artery stenosis was created without inducing a concomitant injury to the arterial wall, cyclic flow reductions could not be spontaneously induced. Yet, infusion of an enzymatic system that generates both superoxide anion and hydrogen peroxide promptly induced cyclic flow reductions [29]. However, it is also possible that those thrombogenic effects were related to oxidant-mediated injury of the endothelial cell layer (as suggested by histological examination of the vessels), rather than to a direct stimulating effect on platelets. A possible inhibitory role of exogenous hydrogen peroxide on platelet aggregation/thrombus formation in vivo is suggested by observations performed in our laboratory. In a rabbit model of carotid artery stenosis and endothelial injury, similar to the canine model previously described, cyclic flow reductions spontaneously developed at the site of endothelial injury. These spontaneous episodes of intravascular platelet aggregation could be abolished in all animals by local infusion of H2O2 [31] (Fig. 3).


Figure 3
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Effects of hydrogen peroxide infusion on blood flow in rabbit carotid arteries with a stenosis and endothelial injury. Data are from [31]. Open bars represent baseline values. Hatched bars are data obtained during intracarotid infusion of hydrogen peroxide (average dose 6 µmol/min). Solid bars are data obtained 10 min into washout. Left panel: Effects of hydrogen peroxide on the number of episodes of flow reduction, measured as cycles/hour. Right panel: Effects of hydrogen peroxide on peak carotid flow, measured as percent of normal values. Note that H2O2 infusion abolished cyclic flow reductions (caused by intravascular platelet thrombus formation) and restored normal flow. This antiaggregatory effect was lost on washout. *P<0.05 vs. baseline and washout.

 
Taken together, the in vivo data are consistent with two major observations obtained in in vitro platelet studies, namely that low levels of oxidants may promote aggregation [12, 16–21] whereas exposure to high concentrations of exogenous H2O2 may result in platelet inhibition [13–15, 21]. However, the in vivo situation is clearly more complex, since the net effect of oxidants on intravascular thrombosis is also dependent on the integrity of the endothelium, as well as on oxidant-mediated alterations of other major players of thrombosis, such as endothelial-derived relaxing factor and coagulation factors. These issues will also be briefly reviewed.


   3 Effects of oxidants on the vessel wall
Top
 Abstract
 1 Characteristics of reactive...
 2 Effects of oxidants...
 3 Effects of oxidants...
4 Effects of oxidants...
 5 Effects of oxidants...
 6 Leukocytes and thrombus...
 7 Conclusions
 References
 
Under physiologic conditions the vascular endothelium plays a pivotal role in inhibiting intravascular thrombus formation, since a dynamic interaction exists at the endothelial cell surface between mechanisms which respectively inhibit or promote thrombus formation. Endothelial cells can inhibit intravascular thrombosis by synthesizing various substances, such as thrombomodulin, tissue factor-pathway inhibitor, prostacyclin, tissue plasminogen activator [32], which inhibit the coagulation cascade and platelet function, or which activate fibrinolysis. Disruption of endothelial integrity by oxidant attack will grossly impair these activities. However, oxidants may also influence the anti-thrombotic properties of the endothelium in a more specific way, even at relatively low concentrations. This may happen through interaction with components of the coagulation system (as described in the next section), or with endothelial-derived relaxing factor (EDRF).

Endothelial cells release EDRF also in the vascular lumen. In vitro, EDRF has been shown to exert antiplatelet effects via stimulation of guanylate cyclase [25]. That endogenous EDRF is important in counteracting platelet aggregation has been directly demonstrated in vivo by studies in which stimulation of EDRF release by intra-arterial infusion of acetylcholine inhibited intravascular thrombus formation at sites of arterial stenosis and endothelial injury [33, 34]. Furthermore, in one of these studies we were able to show that administration of L-NMMA (an inhibitor of endogenous EDRF production) to animals in which cyclic flow reductions had been previously abolished with aspirin was associated with restoration of cyclic flow reductions [33], thus demonstrating the importance of EDRF in modulating intravascular platelet aggregation in vivo. There are reasons to believe that EDRF could be another potential target for the effects of oxidants.

Superoxide anions have been implicated in the breakdown of EDRF. In vitro, the half-life of EDRF is significantly shortened in the presence of superoxide radicals and, conversely, superoxide dismutase has been demonstrated to prolong EDRF half-life [35, 36]. Indirect support to the involvement of oxygen radicals in reducing the effects of EDRF in vivo comes from the finding that formation of active oxygen species during reperfusion of ischemic myocardium causes vessel dysfunction and that superoxide dismutase protects the vasodilator ability of coronary vessels [37], and from the observation that release of active EDRF depends on preserved endothelial superoxide dismutase activity [38]. Taken together, these findings would suggest that active oxygen species might facilitate intravascular thrombus formation by reducing the antiplatelet effects of EDRF.


   4 Effects of oxidants on the coagulation cascade
Top
 Abstract
 1 Characteristics of reactive...
 2 Effects of oxidants...
 3 Effects of oxidants...
 4 Effects of oxidants...
5 Effects of oxidants...
 6 Leukocytes and thrombus...
 7 Conclusions
 References
 
As previously stated, under normal conditions endothelial cells synthesize a variety of substances which may inhibit intravascular thrombus formation. In addition, since the endothelium is in contact with circulating blood, synthesis and expression of one important component of the coagulation pathway, ‘tissue factor’ (TF), is normally suppressed in these cells [32]. The result of these regulatory mechanisms is a tight control of the coagulation system, such that unwanted intravascular thrombus formation is normally inhibited. TF is a 47-kDa membrane-bound glycoprotein essential for activation of the extrinsic coagulation pathway. TF forms a complex with coagulation factors VII and VIIa, allowing enzymatic activation of factors X and IX, the substrates for factor VIIa [39], ultimately leading to the generation of thrombin. The importance of TF in triggering intravascular thrombus formation in vivo has been directly shown in a recent study in which endothelial disruption at the site of arterial stenosis, with its attendant exposure of TF present in the subendothelium, resulted in intravascular thrombus formation via direct activation of the extrinsic coagulation pathway [40].

To protect against this unwanted intravascular activation of the coagulation system, normal endothelium lacks TF activity [32, 41]. However, several stimuli can affect anticoagulant properties of the endothelium by inducing TF expression on the membrane of endothelial cells [42, 43]. Endothelial cells represent both a source and a possible target of oxidants released in the vasculature [44]. At the same time, oxidants are known to activate nuclear transcription factors [45]. Thus, we felt it was important to determine whether oxygen radicals would also promote de novo synthesis of TF activity by endothelial cells. To test this hypothesis, endothelial cells were exposed to exogenously generated oxygen radicals. A brief period of exposure to oxygen radicals resulted in a significant increase in TF mRNA levels, accompanied by appearance of large TF procoagulant activity [46] (Fig. 4). This phenomenon was not confined to endothelial cells in vitro. In rabbits subjected to coronary artery occlusion and reperfusion, a condition associated with endogenous production of large amounts of oxygen radicals, we detected a marked increase in tissue factor activity in the coronary circulation [46]. Importantly, this phenomenon was accompanied by a significant reduction of myocardial perfusion, and it could be abolished by oxygen radical scavengers [46]. We speculate that this oxygen radical-mediated TF expression by endothelial cells, with its attendant activation of the extrinsic coagulation pathway, may have important consequences as it might impact on the pathophysiology of post-ischemic reperfusion.


Figure 4
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Effects of oxidants on tissue factor mRna levels and activity in cultured endothelial cells (data are from [46]). Oxidants were generated by 5 min incubation with xanthine/xanthine oxidase (X/XO; 400 µM and 20 mU/ml, respectively). Left panel: Northern blot of RNA isolated from control (left) and oxygen radical-treated (right) cells. TF mRNA was almost undetectable in control cells, but it markedly increased following exposure to oxygen radicals. Glyceraldehyde-phosphate dehydrogenase (GADPH) mRNA was used as internal control. Right panel: TF activity, measured by the amount of factor Xa generated. TF activity was almost undetectable in controls cells. Incubation of cells with X/XO for 1 or 5 min resulted in a marked increase in TF activity. Cells simultaneously incubated with AP-1, a monoclonal antibody against TF, showed values similar to controls. ND=undetectable. *P<0.05 vs. controls.

 

   5 Effects of oxidants on anti-coagulant factors
Top
 Abstract
 1 Characteristics of reactive...
 2 Effects of oxidants...
 3 Effects of oxidants...
 4 Effects of oxidants...
 5 Effects of oxidants...
6 Leukocytes and thrombus...
 7 Conclusions
 References
 
In addition to inducing TF expression in endothelial cells, active oxygen species might promote intravascular thrombus formation also by interfering with mechanisms that normally inhibit activation of the coagulation pathway. Earlier studies have shown that lipid peroxides can increase the amount of thrombin produced and can slow down the rate of thrombin decay [47]. Both effects are consequent to inhibition of plasma antithrombin by lipid peroxides formed as a consequence of oxygen radical attack to circulating lipoproteins [48]. Similar susceptibility to oxidant-mediated inactivation has been reported for other key anti-thrombotic factors, such as alpha-2-antiplasmin [49], plasminogen activator [50], and thrombomodulin [51]. More recently, in a preliminary study we have shown that endothelial cell exposed to oxygen radicals exhibit, in addition to the induction of TF procoagulant activity, a concomitant marked decrease of tissue factor-pathway inhibitor (TFPI) activity to almost undetectable levels [52]. TFPI is a protein synthesized by endothelial cells that inhibits the extrinsic coagulation pathway.


   6 Leukocytes and thrombus formation
Top
 Abstract
 1 Characteristics of reactive...
 2 Effects of oxidants...
 3 Effects of oxidants...
 4 Effects of oxidants...
 5 Effects of oxidants...
 6 Leukocytes and thrombus...
7 Conclusions
 References
 
It has long been known that leukocytes could be found within thrombi. However, the prevailing theory was that leukocytes take no part in the coagulation process, but they are merely trapped by the polymerization of fibrin. This ‘mechanical’ theory to explain the association of leukocytes with thrombi has recently been challenged. In an elegant experiment, Palabrica et al. [53] demonstrated that leukocyte accumulation within thrombi is in fact a cellular adhesion-mediated event. According to the results of that study, platelets which accumulate in the thrombus rapidly express the adhesion molecule P-selectin on their surface, thereby inducing adherence of circulating monocytes and neutrophils. This would lead to a chain of events culminating in tissue factor-mediated local fibrin deposition.

The study by Palabrica et al. [53] was concerned with the interaction between leukocytes and platelets already trapped in the thrombus. However, these two cell types can adhere and ‘cross-talk’ to each other also under dynamic conditions, both in vitro [54] and in vivo [55]. The net effect of this phenomenon on the coagulation system is difficult to predict, as certain secretory products of activated neutrophils can inhibit platelet aggregation (e.g., hydrogen peroxide [13–15, 21], nitric oxide [25], lipoxygenase metabolites [56]), while others have stimulatory effects (PAF [27], cathepsin G [57]). However, it is possible that under certain conditions intravascular leukocyte activation may contribute to thrombus formation. In the clinical setting, leukocyte-platelet aggregates have been found in the blood of patients with unstable angina [55], and it has been suggested that activation of leukocytes may contribute to intravascular coagulation in patients with septicemia [58].


   7 Conclusions
Top
 Abstract
 1 Characteristics of reactive...
 2 Effects of oxidants...
 3 Effects of oxidants...
 4 Effects of oxidants...
 5 Effects of oxidants...
 6 Leukocytes and thrombus...
 7 Conclusions
References
 
In summary, reactive oxygen metabolites might affect thrombus formation within the vasculature through several mechanisms. Oxidants may enhance the activity of the extrinsic coagulation cascade, ultimately leading to thrombin formation, via their combined effects on stimulation of tissue factor activity and inhibition of fibrinolytic pathways. At the same time, oxidants may have complex effects of platelets, since while hydrogen peroxide acts to inhibit aggregation superoxide radical may inactivate EDRF and PAF-acetylhydrolase, and it may also enhance thrombin formation (via TF), all events which would promote aggregation. The net result of all these effects may depend on a number of factors, the relative importance of which is not easily dissected out and will require much further investigation. Similarly, further studies are warranted to investigate whether the oxidant load which may occur upon postischemic reflow might contribute at least in part to the high incidence of reocclusion frequently seen in patients with acute myocardial infarction undergoing thrombolytic therapy, and whether enhanced oxidative stress is part of the picture of patients with unstable angina.

Time for primary review 28 days.


   Acknowledgements
 
Supported in part by grant No. 96.03489.CT04, from Consiglio Nazionale delle Ricerche, Italy.


   References
Top
 Abstract
 1 Characteristics of reactive...
 2 Effects of oxidants...
 3 Effects of oxidants...
 4 Effects of oxidants...
 5 Effects of oxidants...
 6 Leukocytes and thrombus...
 7 Conclusions
 References
 

  1. Fridovich I. The biology of oxygen radicals. Science (1978) 201:6049–6055.
  2. Ambrosio G., Zweier J.L., Duilio C., Kuppusamy P., Santoro G., Elia P.P., Tritto I., Cirillo P., Condorelli M., Chiariello M., Flaherty J.T. Evidence that mitochondrial respiration is a source of potentially toxic oxygen free radicals in intact rabbit hearts subjected to ischemia and reflow. J. Biol. Chem. (1993) 268:18532–18541.[Abstract/Free Full Text]
  3. Kukreja R.K., Kontos H.A., Hess M.L., Ellis E.F. PGH synthase and lipoxygenase generate superoxide in the presence of NADH or NADPH. Circ. Res. (1986) 59:612–619.[Abstract/Free Full Text]
  4. Fantone J.C., Ward P.A. Role of oxygen-derived free radicals and metabolites in leucocyte-dependent inflammatory reactions. Am. J. Pathol. (1982) 107:397–418.[Web of Science]
  5. Zweier J.L., Flaherty J.T., Weisfeldt M.L. Direct measurements of free radical generation following reperfusion of ischemic myocardium. Proc. Natl. Acad. Sci. USA (1987) 84:1404–1407.[Abstract/Free Full Text]
  6. Bolli R., Patel B.S., Jeroudi M.O., Lai E.K., McCay P.B. Demonstration of free radical generation in ‘stunned’ myocardium of intact dogs with the use of the spin trap alpha-phenyl N-tert-butyl nitrone. J. Clin. Invest. (1988) 82:476–485.[Web of Science][Medline]
  7. McCord J.M. Oxygen-derived free radicals in post-ischemic tissue injury. New Engl. J. Med. (1985) 312:159–163.[Abstract]
  8. Halliwell B. Oxidants and human disease: some new concepts. FASEB J. (1987) 1:358–364.[Abstract]
  9. Ambrosio G., Flaherty J.T., Duilio C., Tritto I., Santoro G., Elia P.P., Condorelli M., Chiariello M. Oxygen radicals generated at reflow induce peroxidation of membrane lipids in reperfused hearts. J. Clin. Invest. (1991) 87:2056–2066.[Web of Science][Medline]
  10. McCord J.M. Superoxide radical: controversies, contradictions, and paradoxes. Proc. Soc. Exp. Biol. Med. (1995) 209:112–117.[Abstract]
  11. Finazzi-Agro' A., Menichelli A., Persiani M., Biancini G., Del Principe D. Hydrogen peroxide release from human blood platelets. Biochim. Biophys. Acta (1992) 718:21–25.
  12. Del Principe D., Menichelli A., De Matteis W., Di Giulio S., Giordani M., Savini I., Finazzi-Agro' A. Hydrogen peroxide is an intermediate in the platelet activation cascade triggered by collagen, but not by thrombin. Thromb. Res. (1991) 62:365–375.[CrossRef][Web of Science][Medline]
  13. Canoso R.T., Rodvien R., Scoon K., Levine P.H. Hydrogen peroxide and platelet function. Blood (1974) 43:645–656.[Abstract/Free Full Text]
  14. Levine P.H., Weinger R.S., Simon J., Scoon K.L., Krinsky N.I. Leucocyte-platelet interaction. Release of hydrogen peroxide by granulocytes as a modulator of platelet reactions. J. Clin. Invest. (1976) 57:955–963.[Web of Science][Medline]
  15. Stuart M.J., Holmsen H. Hydrogen peroxide, an inhibitor of platelet function: effect on adenine nucleotide metabolism, and the release reaction. Am. J. Hematol. (1977) 2:53–63.[Web of Science][Medline]
  16. Del Principe D., Menichelli A., De Matteis W., Di Corpo M.L., Di Giulio S., Finazzi-Agro' A. Hydrogen peroxide has a role in the aggregation of human platelets. FEBS Lett. (1985) 185:142–146.[CrossRef][Web of Science][Medline]
  17. Praticò D., Iuliano L., Alessandri C., Camastra C., Violi F. Polymorphonuclear leukocyte-derived O2-reactive species activate primed platelets in human whole blood. Am. J. Physiol. (1993) 264:H1582–H1587.[Web of Science][Medline]
  18. Handin R.I., Karabin R., Boxer G.J. Enhancement of platelet function by superoxide anion. J. Clin. Invest. (1977) 59:959–965.[Web of Science][Medline]
  19. Salvemini D., De Nucci G., Sneddon M., Vane J.R. Superoxide anions enhance platelet adhesion and aggregation. Br. J. Pharmacol. (1989) 97:1145–1150.[Web of Science][Medline]
  20. Salvemini D., De Nucci G., Vane J.R. Superoxide dismutase cooperates with prostacyclin to inhibit platelet aggregation: a comparative study in washed platelets and platelet rich plasma. Thromb. Haemost. (1991) 65:421–424.[Web of Science][Medline]
  21. Ambrosio G., Golino P., Pascucci I., Rosolowsky M., Campbell W.B., DeClerck F., Tritto I., Chiariello M. Modulation of platelet function by reactive oxygen metabolites. Am. J. Physiol. (1994) 267:H308–H318.[Web of Science][Medline]
  22. Suematsu M., Schmid-Schönbein G.W., Chavez-Chavez R.H., Yee T.T., Tamatani T., Miyasaka M., Delano F.A., Zweifach B.W. In vivo visualization of oxidative changes in microvessels during neutrophil activation. Am. J. Physiol. (1993) 264:H881–H891.[Web of Science][Medline]
  23. Burke T.M., Wolin M.S. Hydrogen peroxide elicits pulmonary arterial relaxation and guanylate cyclase activation. Am. J. Physiol. (1987) 252:H721–H732.[Web of Science][Medline]
  24. Stamler J.S., Vaughan D.E., Loscalzo J. Synergistic disaggregation of platelets by tissue-type plasminogen activator, prostaglandin E1, and nitroglycerin. Circ. Res. (1988) 63:1070–1079.[Abstract/Free Full Text]
  25. Mellion B.T., Ignarro L.J., Ohlstein E.H., Pontecorvo E.G., Hyman A.L., Kadowitz P.J. Evidence for the inhibitory role of guanosine 3',5'-monophosphate in ADP-induced human platelet aggregation in the presence of nitric oxide and related nitrovasodilators. Blood (1981) 57:946–955.[Free Full Text]
  26. Hemler M.E., Lands W.E.M. Evidence for a peroxide-initiated free radical mechanism of prostaglandin biosynthesis. J. Biol. Chem. (1980) 255:6253–6261.[Abstract/Free Full Text]
  27. Ambrosio G., Oriente A., Napoli C., Palumbo G., Chiariello P., Marone G., Condorelli M., Chiariello M., Triggiani M. Oxygen radicals inhibit human plasma acetylhydrolase, the enzyme that catabolizes platelet-activating factor. J. Clin. Invest. (1994) 93:2408–2416.[Web of Science][Medline]
  28. Kubes P., Suzuki M., Granger D.N. Modulation of PAF-induced leukocyte adherence and increased microvascular permeability. Am. J. Physiol. (1990) 259:G859–G864.[Web of Science][Medline]
  29. Yao S.K., Ober J.C., Gonenne A., Clubb F.J., Krishnaswami A., Ferguson J.J., Anderson V.H., Gorecki M., Buja L.M., Willerson J.T. Active oxygen species play a role in mediating platelet aggregation and cyclic flow variations in severely stenosed and endothelium-injured coronary arteries. Circ. Res. (1993) 73:952–967.[Abstract/Free Full Text]
  30. Ikeda H., Koga Y., Oda T., Kuwano K., Nakayama H., Ueno T., Toshima H., Michael L.H., Entman M.L. Free oxygen radicals contribute to platelet aggregation and cyclic flow variations in stenosed and endothelium-injured canine coronary arteries. J. Am. Coll. Cardiol. (1994) 24:1749–1756.[Abstract]
  31. Golino P., Ambrosio G., Ragni M., Russolillo E., Focaccio A., Chiariello M. Hydrogen peroxide inhibits cyclic flow reductions in rabbit stenotic carotid arteries with endothelial injury [abstract]. Eur. Heart J. (1992) 12(Suppl.):299.
  32. Jaffe EA. Endothelial cell structure and function. In: Hoffman R, Benj EJ, Shattil SJ, Furie B, Cohen HJ, eds. Hematology: Basic Principles and Practice. New York: Churchill Livingstone, 1991:1198–1215.
  33. Golino P., Cappelli-Bigazzi M., Ambrosio G., Ragni M., Russolillo E., Condorelli M., Chiariello M. Endothelium-derived relaxing factor modulates platelet aggregation in an in vivo model of recurrent platelet activation. Circ. Res. (1992) 71:1447–1453.[Abstract/Free Full Text]
  34. Yao S.K., Ober J., Krishnaswami A., Ferguson J.J., Anderson H.V., Golino P., Buja L.M., Willerson J.T. Endogenous endothelium-derived relaxing factor protects against platelet aggregation and cyclic flow variations in stenosed and endothelium-injured arteries. Circulation (1992) 86:1302–1310.[Abstract/Free Full Text]
  35. Rubanyi G.M., Vanhoutte P.M. Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor. Am. J. Physiol. (1986) 250:H822–H827.[Web of Science][Medline]
  36. Gryglewski R.J., Palmer R.M.J., Moncada S. Superoxide anion is involved in the breakdown of endothelium-derived relaxing factor. Nature (1986) 320:454–456.[CrossRef][Medline]
  37. Mehta J.L., Nichols W.W., Donnelly W.H., Lawson D.L., Thompson L., Ter Riet M., Salden T.G.P. Protection by superoxide dismutase from myocardial dysfunction and attenuation of vasodilator reserve after coronary occlusion and reperfusion in dog. Circ. Res. (1989) 65:1283–1295.[Abstract/Free Full Text]
  38. Mugge A., Elwell J.H., Peterson T.E., Harrison D.G. Release of intact endothelium-derived relaxing factor depends on endothelial superoxide dismutase activity. Am. J. Physiol. (1991) 260:C219–C225.[Web of Science][Medline]
  39. Rapaport S.I., Rao L.V.M. Initiation and regulation of tissue factor-dependent blood coagulation. Arterioscler. Thromb. (1992) 12:1111–1121.[Medline]
  40. Pawashe A.B., Golino P., Ambrosio G., Migliaccio F., Ragni M., Pascucci I., Chiariello M., Bach R., Garen A., Konigsberg W.K., Ezekowitz M.D. A monoclonal antibody against rabbit tissue factor inhibits thrombus formation in stenotic injured rabbit carotid arteries. Circ. Res. (1994) 74:56–63.[Abstract/Free Full Text]
  41. Wilcox J.N., Smith K.M., Schwartz S., Gordon D. Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque. Proc. Natl. Acad. Sci. USA (1989) 86:2839–2843.[Abstract/Free Full Text]
  42. Crossman D.C., Carr D.P., Tuddenham E.G.D., Pearson J.D., McVey J.H. The regulation of tissue factor mRNA in human endothelial cells in response to endotoxin or phorbol ester. J. Biol. Chem. (1990) 265:9782–9787.[Abstract/Free Full Text]
  43. Kao J. Endothelial monocyte-activating polypeptide II. A novel tumor-derived polypeptide that activates host-response mechanisms. J. Biol. Chem. (1992) 267:20239–20247.[Abstract/Free Full Text]
  44. Zweier J.L., Broderick R., Kuppusamy P., Thompson-Gorman S., Lutty G.A. Determination of the mechanism of free radical generation in human aortic endothelial cells exposed to anoxia and reoxygenation. J. Biol. Chem. (1994) 269:24156–24162.[Abstract/Free Full Text]
  45. Rao G.N., Berk B.C. Active oxygen species stimulate vascular smooth muscle cell growth and proto-oncogene expression. Circ. Res. (1992) 70:593–599.[Abstract/Free Full Text]
  46. Golino P., Ragni M., Cirillo P., Avvedimento V.E., Feliciello A., Esposito N., Scognamiglio A., Trimarco B., Iaccarino G., Condorelli M., Chiariello M., Ambrosio G. Effects of tissue factor induced by oxygen free radicals on coronary flow during reperfusion. Nature Med. (1996) 2:35–40.[CrossRef][Web of Science][Medline]
  47. Barrowcliffe T.W., Gutteridge J.M.C., Dormann T.L. The effects of fatty acid autoxidation products on blood coagulation. Thromb. Diath. Haemorrh. (1975) 23:271–277.
  48. Gray E., Barrowcliffe T.W. Inhibition of antithrombin III by lipid peroxides. Thromb. Res. (1985) 37:241–250.[CrossRef][Web of Science][Medline]
  49. Stief T.W., Aab A., Heimburger H. Oxidative inactivation of purified human alpha-2-antiplasmin, antithrombin III, and C1-inhibitor. Thromb. Res. (1988) 49:581–589.[CrossRef][Web of Science][Medline]
  50. Lawrence D.A., Loskutoff D.J. Inactivation of plasminogen activator inhibitor by oxidants. Biochemistry (1986) 25:6351–6355.[CrossRef][Web of Science][Medline]
  51. Glaser C.B., Morser J., Clarke J.H., Blasko E., McLean K., Kuhn I., Chang R.J., Lin J.H., Vilander L., Andrews W.H., Light D.R. Oxidation of a specific methionine in thrombomodulin by activated neutrophil products blocks cofactor activity. A potential rapid mechanism for modulation of coagulation. J. Clin. Invest. (1992) 90:2565–2573.[Web of Science][Medline]
  52. Golino P., Ambrosio G., Ragni M., Cirillo P., Scognamiglio A., Condorelli M., Chiariello M. Oxygen radicals induce a procoagulant state in cultured coronary endothelial cells by inducing tissue factor synthesis and by inhibiting tissue-factor pathway inhibitor [abstract]. Circulation (1995) 92:I354.
  53. Palabrica T., Lobb R., Furie B.C., Aronovitz M., Benjamin C., Hsu Y.M., Sajer S.A., Furie B. Leukocyte accumulation promoting fibrin deposition is mediated in vivo by P-selectin on adherent platelets. Nature (1992) 359:848–851.[CrossRef][Medline]
  54. Evangelista V., Manarini S., Rotondo S., Martelli N., Polishuck R., McGregor J.L., De Gaetano G., Cerletti C. Platelet/polymorphonuclear leukocyte interaction in dynamic conditions: evidence of adhesion cascade and cross talk between P-selectin and the β2 integrin CD11b/CD18. Blood (1996) 88:4183–4194.[Abstract/Free Full Text]
  55. Ott I., Neumann F.-J., Gawaz M., Schmitt M., Schomig A. Increased neutrophil-platelet adhesion in patients with unstable angina. Circulation (1996) 94:1239–1246.[Abstract/Free Full Text]
  56. Valles J., Santos M.T., Marcus A.J., Safier L.B., Broekman M.J., Islam N., Ullman H.L., Aznar J. Downregulation of human platelet reactivity by neutrophils. Participation of lipooxygenase derivatives and adhesive proteins. J. Clin. Invest. (1993) 92:1357–1365.[Web of Science][Medline]
  57. Del Maschio A., Evangelista V., Rajtar G., Chen Z.M., Cerletti C., De Gaetano G. Platelet activation by polymorphonuclear leukocytes exposed to chemotactic agents. Am. J. Physiol. (1990) 258:H870–H879.[Web of Science][Medline]
  58. Okajima K., Yang W.P., Okabe H., Inoue M., Takatsuki K. Role of leukocytes in the activation of intravascular coagulation in patients with septicemia. Am. J. Hematol. (1991) 36:265–271.[Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Ambrosio, G.
Right arrow Articles by Golino, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ambrosio, G.
Right arrow Articles by Golino, P.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?