(Circulation. 1997;95:787-789.)
© 1997 American Heart Association, Inc.
Articles |
Reactive Oxygen Species and Platelet Activation in Reperfusion Injury
the Centre for Cardiovascular Science, Royal College of Surgeons in Ireland, St. Stephens Green, Dublin.
Correspondence to Desmond J. Fitzgerald, MD, Centre for Cardiovascular Science, Royal College of Surgeons in Ireland, St. Stephens Green, Dublin 2, Ireland. E-mail dfitzgerald@rcsi.ie.
Key Words: Editorials • platelets • reperfusion • free radicals
 |
Introduction |
|---|
 Top Introduction Consequences of OFR Production...OFRs and Platelet ActivationOFRs Have Indirect Effects...References |
|---|
Reperfusion of ischemic myocardium results in an abrupt aggravation of the cardiomyocyte injury. Compelling evidence that cell injury results from reoxygenation during reperfusion has been demonstrated experimentally by the introduction of oxygen into hypoxic hearts, resulting in abrupt ultrastructural damage to the surrounding tissue.1 This reperfusion/reoxygenation injury appears to be due to the generation of OFRs, a highly reactive species that interact with several cellular targets and result in injury. Electron spin resonance spectroscopy and spin trapping agents have demonstrated increases in the production of OFRs during ischemia/reperfusion,2 3 and the use of antioxidants and free radical scavengers has implicated OFRs as important mediators of the subsequent cell injury.
|
Consequences of OFR Production in Vivo |
|---|
|
TopIntroductionConsequences of OFR Production...OFRs and Platelet ActivationOFRs Have Indirect Effects...References |
|---|
There are several potential sources of OFR generation (see the Table
) during ischemia/reperfusion of vascular tissue.4 5 6 Platelets can produce superoxide (O2-), as can almost all aerobic cells, but the primary source after an oxygen burst is via neutrophil NADPH oxidase or from leakage of electrons from the electron transport chain in the mitochondria.7 Most of the O2- anions produced escape into the surrounding medium and form H2O2 through the activity of superoxide dismutase. H2O2 is not a free radical but can react with O2- and generate the highly reactive hydroxyl radical OH·. Another potential source of free radicals is arachidonic acid metabolism by cyclooxygenase and lipooxygenase, which generate intermediate peroxy compounds, hydroperoxy compounds, and OH· radicals.8
|
All the major classes of biomolecules can be attacked by oxygen free radicals, so their deleterious effects are wide ranging. Particularly susceptible are polyunsaturated fatty acids in which oxidation generates reactive lipid peroxides (ROO·) and a self-perpetuating chain reaction of lipid peroxidation. Oxidation of membrane phospholipids may disrupt the membrane integrity and fluidity and perhaps interfere with the function of receptors on the cell surface. Recently, a novel series of products with biological function have been identified that are free radical–derived products of arachidonic acid and other polyunsaturated fatty acids.9 Called isoprostanes because of their structural similarity to classic prostaglandins, these compounds activate platelets and vascular smooth muscle cells through a receptor that is similar but not identical to the TxA2 receptor.10 Isoprostanes are largely generated and remain esterified within the cell membrane. However, some products can be detected in urine and have been used as markers of free radical–induced cell injury in vivo.11 DNA and several proteins are also vulnerable targets for oxidative damage. In addition, free radical–induced injury can trigger the expression of several genes, including those regulating programmed cell death. Indeed, this process has been demonstrated in a rabbit model of myocardial reperfusion that used in situ labeling to detect the characteristic DNA fragmentation pattern of programmed cell death in cardiomyocytes.12
The cardiomyocyte is not the only cell attacked by OFRs during coronary reperfusion. Endothelial cell injury has long been recognized to occur, manifesting as a loss of endothelium-dependent relaxation and an abrupt loss of thromboresistance. The latter may reflect several mechanisms, including inactivation of cyclooxygenase and thrombomodulin, the latter resulting from oxidation of a methionine in the thrombin-binding domain.13 The paper by Leo and colleagues14 in this issue of Circulation suggests that platelets may also be modified after reoxygenation. Studies in humans and experimental models have shown a burst of platelet activation immediately on coronary reperfusion.15 The increased platelet activity provides the substrate for continued thrombosis and coronary reocclusion. Although several agonists, including TxA2 and thrombin, have been implicated, the primary mediator of platelet activation after coronary reperfusion has not been identified. Leo and colleagues show spontaneous platelet aggregation on reoxygenation of anoxic platelets and suggest that this is due to the generation of OFRs.
|
OFRs and Platelet Activation |
|---|
|
TopIntroductionConsequences of OFR Production...OFRs and Platelet ActivationOFRs Have Indirect Effects...References |
|---|
Previous reports by this group have shown that H2O2 can trigger the activation of platelets "primed" with subthreshold concentrations of arachidonic acid or agonists such as collagen that transduce a signal mediated by arachidonic acid metabolism.16 Platelet activation has also been demonstrated with superoxide dismutase, presumably by its ability to produce H2O2.17 Activation of primed platelets in these circumstances may occur through PLA2-induced release of arachidonic acid and subsequent generation of TxA2, as aspirin and mepacrine (a PLA2 inhibitor) were shown to block the effect.18 Platelet activation by H2O2 was shown to be prevented by OH· radical scavengers and iron chelators, suggesting that the responsible species is OH· formed from H2O2 in a Fenton-like reaction. Agonist-induced platelet activation may be mediated in part by endogenously generated OH· radicals, particularly when the response involves the release of arachidonic acid.19 Platelet agonists stimulate the production of O2- by NADPH oxidase and the release of arachidonic acid by PLA2. O2- then dismutes to H2O2, giving rise to OH· radicals. Arachidonic acid scavenging of OH· radicals leads to the formation of a peroxyl radical, a potent activator of cyclooxygenase.
In this issue of Circulation, Leo and coworkers14 provide evidence that platelet activation occurs after reoxygenation of anoxic platelets, and their findings implicate a role for OFRs because platelet aggregation on reoxygenation was significantly reduced in the presence of OFR scavengers. Moreover, there was a time-dependent increase in the release of both OH· and O2- radicals from platelets exposed to anoxia/reoxygenation. A number of potential sources for the production of OFRs in platelets were examined. The inhibition of NADPH oxidase almost completely inhibited O2- release, as did aspirin, suggesting that cyclooxygenase activity was also a major source of the OFRs. Curiously, much of the response was also mediated by cyclooxygenase activity through the metabolism of endogenous arachidonic acid to TxA2. The increase in TxA2 formation and subsequent activation of its receptor may have been responsible for many of the intracellular signaling detected, including the activation of phospolipase C and PLA2 and secondary release of endogenous arachidonic acid. Thus, it is still unclear which OFRs are generated by reoxygenation alone or how this initiates platelet activation and secondary TxA2 formation.
|
OFRs Have Indirect Effects on Platelet Activation |
|---|
|
TopIntroductionConsequences of OFR Production...OFRs and Platelet ActivationOFRs Have Indirect Effects...References |
|---|
In addition to a direct effect, OFRs may influence platelets in vivo by modifying the synthesis, release, and activity of many mediators that modulate their activity. Endothelium-derived NO is one such mediator that can alter platelet function and is itself sensitive to the oxidant status of the surrounding environment. NO is a potent vasodilator and inhibitor of platelet aggregation. It inhibits aggregation through generation of intracellular cGMP, which inhibits subsequent platelet responses to agonists.20 21 NO can scavenge superoxide, rendering it inactive and forming nitrite. However, this reaction can also form a peroxynitrite anion (ONOO-) that can give rise to OH· and nitrogen dioxide radicals when protonated, both of which may damage tissues. ONOO- has been shown to have direct but paradoxical effects on platelets, depending on the surrounding environment.22 ONOO- can reverse the inhibition of platelet aggregation induced by prostacyclin and has both proaggregatory and antiaggregatory functions in platelet-rich plasma, depending on the degradation products formed.22 Another potent vasodilator and inhibitor of platelet aggregation is PGI2. PGI2 and NO act synergistically to inhibit platelet activation and adherence to vessel walls.23 The formation of PGI2 is reduced after reoxygenation of anoxic endothelium because of inactivation of cyclooxygenase by OFRs.24 Thus, impaired production and/or inactivation of PGI2 or NO as a result of endothelial cell oxidant damage could augment the platelet activity occurring during anoxia/reoxygenation.
Reperfusion injury of vascular endothelium may also promote generation of thrombin, which is a major agonist of platelets after coronary reperfusion. The activity of thrombin is highly regulated in part by binding to thrombomodulin on the surface of endothelial cells. Binding to thrombomodulin alters the substrate specificity of thrombin so that it loses its procoagulant and platelet activity and activates protein C. Thrombomodulin is highly sensitive to OFRs, which oxidize a critical methionine in the thrombin binding region. An additional platelet agonist whose activity may be enhanced by OFRs is PAF, which also has potent proinflammatory effects.25 PAF is inactivated by PAF-acetylhydrolase, an enzyme that circulates bound to LDL. OFRs can rapidly and irreversibly inactivate PAF-acetylhydrolase.26 Moreover, OFRs induce expression of PAF by vascular endothelial cells.
In conclusion, OFRs generated during reperfusion of hypoxic tissue modify the behavior of several cells, including platelets, that may promote thrombosis and subsequent reocclusion. If this is the case, suppression of OFRs during coronary or cerebral reperfusion may provide a novel approach to preventing vascular reocclusion and a safer alternative to systemic antithrombotic therapy.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This work was supported by grants from the Health Research Board of Ireland, The Irish Heart Foundation, and the Royal College of Surgeons of Ireland.
|
Footnotes |
|---|
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
|
References |
|---|
|
TopIntroductionConsequences of OFR Production...OFRs and Platelet ActivationOFRs Have Indirect Effects...References |
|---|
1. Hearse DJ, Humphrey SM, Nayler WG, Slade A, Bordu D. Ultrastructural damage associated with reoxygenation of anoxic myocardium. J Mol Cell Cardiol. 1975;7:315-324.[Medline] [Order article via Infotrieve]
2.
Zweier JL. Measurement of superoxide derived free radicals in the reperfused heart: evidence for a free radical mechanism of reperfusion injury. J Biol Chem. 1988;263:1353-1357.
3. Bolli R, Patel BS, Jeroudi MO, Lai EK, McCay PB. 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.
4. McCord JM. Oxygen-derived free radicals in post-ischemic tissue injury. N Engl J Med. 1985;312:159-163.[Abstract]
5. Werns SW, Shea MJ, Lucchesi BR. Free radicals in ischemic myocardial tissue. Free Radic Biol Med. 1985;78:61-82.
6. Simpson PJ, Lucchesi BR. Free radicals in myocardial ischemia and reperfusion injury. J Lab Clin Med. 1987;110:13-30.[Medline] [Order article via Infotrieve]
7. Boveris A. Mitochondrial production of superoxide radical and hydrogen peroxide. Adv Exp Med Biol. 1977;78:1-82.
8.
Rowe GT, Manson NH, Caplan M, Hess ML. Hydrogen peroxide and hydroxyl radical mediation of activated leukocyte depression of cardiac sarcoplasmic reticulum: participation of the cyclooxygenase pathway. Circ Res. 1983;59:612-619.
9.
Morrow JD, Hill KE, Burk RF, Nammour TE, Badr KF, Roberts LJ II. A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalysed mechanism. Proc Natl Acad Sci U S A. 1990;87:9383-9387.
10.
Pratico D, Smyth E, Violi F, FitzGerald GA. Local amplification of platelet function by 8-epi prostaglandin F2a is not mediated by thromboxane receptor isoforms. J Biol Chem. 1996;271:14916-14924.
11. Morrow JD, Roberts LJ II. Quantification of noncyclooxygenase derived prostanoids: a marker of oxidative stress. Free Radic Biol Med. 1991;10:195-200.[Medline] [Order article via Infotrieve]
12. Gottlieb RA, Burleson KO, Kloner RA, Babior BM, Engler RL. Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest. 1994;94:1621-1628.
13. Glaser CB, Morser J, Clarke JH, Blasko E, McLean K, Kuhn I, Chang RJ, Lin JH, Vilander L, Andrews WH. 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.
14.
Leo R, Pratico D, Iuliano L, Pulcinelli FM, Ghiselli A, Pignatelli P, Colavita AR, Fitzgerald GA, Violi F. Platelet activation by superoxide anion and hydroxyl radicals intrinsically generated by platelets that had undergone amoxia and then reoxygenated. Circulation.. 1997;95:885-891.
15.
Kerins DM, Roy L, FitzGerald GA. Fitzgerald DJ. Platelet and vascular function during coronary thrombolysis with tissue-type plasminogen activator. Circulation. 1989;80:1718-1725.
16. Pratico D, Iuliano L, Pulcinelli FM, Bonavita MS, Gazzaniga PP, Violi F. Hydrogen peroxide triggers activation of human platelets selectively exposed to nonaggregating concentrations of arachidonic acid and collagen. J Lab Clin Med. 1992;119:364-370.[Medline] [Order article via Infotrieve]
17. Iuliano L, Practico D, Ghiselli M, Bonavita MS, Violi F. Superoxide dismutase triggers activation of primed platelets. Arch Biochem Biophys. 1991;289:180-183.[Medline] [Order article via Infotrieve]
18. Iuliano L, Pedersen JZ, Practico D, Rotilio G, Violi F. Role of hydroxyl radicals in the activation of human platelets. Eur J Biochem. 1994;221:695-704.[Medline] [Order article via Infotrieve]
19. Iuliano L, Practico D, Bonavita MS, Violi F. Involvement of phospholipase A2 in H2O2-dependent platelet activation. Platelets. 1992;2:87-90.
20. Furlong B, Henderson AH, Lewis MJ, Smith JA. Endothelium-derived relaxing factor inhibits in vitro platelet aggregation. Br J Pharmacol. 1987;90:687-692.[Medline] [Order article via Infotrieve]
21. Alhied U, Frolich J, Forstermann U. Endothelium-derived relaxing factor from cultured human endothelial cells inhibits aggregation of human platelets. Thromb Res. 1987;47:561-566.[Medline] [Order article via Infotrieve]
22.
Moro MA, Darley-Usma VM, Goodwin DA, Read NG, Zamora-Pino R, Feelisch M, Radomski MW, Moncada S. Paradoxical fate and biological action of peroxynitrite on human platelets. Proc Natl Acad Sci U S A. 1994;91:6702-6706.
23. Radomski MW, Palmer RMJ, Moncada S. The anti-aggregating properties of vascular endothelium: interactions between prostacyclin and nitric oxide. Br J Pharmacol. 1987;92:639-646.[Medline] [Order article via Infotrieve]
24.
Hempel SL, Haycraft DL, Hoak JC, Spector AA. Reduced prostacyclin formation after reoxygenation of anoxic endothelium. Am J Physiol. 1990;259:C738-C745.
25.
Demopoulos CA, Pinckard RN, Hanahan DJ. Platelet-activating factor (PAF): evidence for 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphoryl-choline as the active component: a new class of lipid chemical mediators. J Biol Chem. 1980;254:9355-9358.
26. Ambrosio G, Oriente A, Napoli C, Palumbo G, Chiariello P, Marone G, Condrorelli M, Chiariello M, Triggiani M. Oxygen radicals inhibit human plasma acetlyhydrolase, the enzyme that catabolises platelet activation factor. J Clin Invest. 1994;93:2408-2416.
This article has been cited by other articles:
 |
|
 |
|
  S. Massberg, G. Enders, F. C. d. M. Matos, L. I. D. Tomic, R. Leiderer, S. Eisenmenger, K. Messmer, and F. Krombach Fibrinogen Deposition at the Postischemic Vessel Wall Promotes Platelet Adhesion During Ischemia-Reperfusion In Vivo Blood, December 1, 1999; 94(11): 3829 - 3838. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Massberg, G. Enders, R. Leiderer, S. Eisenmenger, D. Vestweber, F. Krombach, and K. Messmer Platelet-Endothelial Cell Interactions During Ischemia/Reperfusion: The Role of P-Selectin Blood, July 15, 1998; 92(2): 507 - 515. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Hishikawa, B. S. Oemar, Z. Yang, and T. F. Luscher Pulsatile Stretch Stimulates Superoxide Production and Activates Nuclear Factor-{kappa}B in Human Coronary Smooth Muscle Circ. Res., November 19, 1997; 81(5): 797 - 803. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||