Affiliation:
1. 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.
Abstract
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 (O
2
–
), 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) A
2
receptor antagonist; by diphenyliodonium, an inhibitor of flavoprotein-dependent enzymes; and by arachidonyl trifluoromethyl ketone, a selective inhibitor of cytosolic phospholipase A
2
. Platelets exposed to A-R markedly generated inositol 1,3,4-trisphosphate and TxA
2
, which were inhibited by incubation of platelets with SOD plus catalase.
Conclusions
This study shows that platelets exposed to A-R intrinsically generated O
2
–
and OH°, which in turn activate arachidonic acid metabolism via phospholipases A
2
and C, and provides further support for the use of antioxidant agents as inhibitors of platelet function in ischemia-reperfusion models.
Publisher
Ovid Technologies (Wolters Kluwer Health)
Subject
Physiology (medical),Cardiology and Cardiovascular Medicine
Cited by
107 articles.
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