(Circulation. 1997;95:885-891.)
© 1997 American Heart Association, Inc.
Articles |
Platelet Activation by Superoxide Anion and Hydroxyl Radicals Intrinsically Generated by Platelets That Had Undergone Anoxia and Then Reoxygenated
the Institute of Clinical Medicine I (R.L., L.I., A.R.C., F.V.) and the Department of Experimental Medicine and Pathology (F.M.P., P.P.), University La Sapienza, Rome, Italy; the Center for Experimental Therapeutics, University of Pennsylvania, Philadelphia (D.P., G.A.F.); and the National Institute of Nutrition (A.G.), Rome, Italy.
Correspondence to Francesco Violi, MD, Institute of Clinical Medicine I, University La Sapienza, Policlinico Umberto I, 00185, Rome, Italy.
Background Platelet activation has been demonstrated in experimental and clinical models of ischemia-reperfusion, but the underlying mechanism is still unclear. We mimicked the ischemia-reperfusion model in vitro by exposing platelets to anoxia-reoxygenation (A-R) and evaluated the role of oxygen free radicals (OFRs), which are usually produced during the reperfusion phase, in inducing platelet activation.
Methods and Results Human platelets were exposed to 15 and 30 minutes of anoxia and then reoxygenated. Compared with control platelets kept in atmospheric conditions, platelets exposed to A-R showed spontaneous platelet aggregation (SPA), which was maximal after 30 minutes of anoxia. Superoxide dismutase (SOD) (–74%, P<.005), catalase (–67%, P<.005), SOD plus catalase (–82%, P<.005), and the hydroxyl radical (OH°) scavengers mannitol (–66%, P<.005) and deoxyribose (–55%, P<.005) inhibited SPA. Platelets that had undergone A-R released superoxide anion (O2–), as detected by lucigenin chemiluminescence. Also, platelets exposed to A-R and incubated with salicylic acid generated 2,3- and 2,5-dihydroxybenzoates, which derive from salicylic acid reaction with OH°. SPA was significantly inhibited by the cyclooxygenase enzyme inhibitors aspirin and indomethacin; by SQ29548, a thromboxane (Tx) A2 receptor antagonist; by diphenyliodonium, an inhibitor of flavoprotein-dependent enzymes; and by arachidonyl trifluoromethyl ketone, a selective inhibitor of cytosolic phospholipase A2. Platelets exposed to A-R markedly generated inositol 1,3,4-trisphosphate and TxA2, which were inhibited by incubation of platelets with SOD plus catalase.
Conclusions This study shows that platelets exposed to A-R intrinsically generated O2– and OH°, which in turn activate arachidonic acid metabolism via phospholipases A2 and C, and provides further support for the use of antioxidant agents as inhibitors of platelet function in ischemia-reperfusion models.
Key Words: platelets • free radicals • hypoxia
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