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(Circulation. 1999;99:3118-3124.)
© 1999 American Heart Association, Inc.

Clinical Investigation and Reports

Iron-Dependent Human Platelet Activation and Hydroxyl Radical Formation

Involvement of Protein Kinase C

Domenico Praticò, MD; Mehtap Pasin, MD; Orla P. Barry, PhD; Andrea Ghiselli, MD; Giuseppe Sabatino, MD; Luigi Iuliano, MD; Garret A. FitzGerald, MD; Francesco Violi, MD

From the Institute of Clinical Medicine I, University La Sapienza (M.P., G.S., L.I., F.V.) and the National Institute of Nutrition (A.G.), Rome, Italy; Department of Medicine (G.S.), University of Chieti, Italy; and the Center for Experimental Therapeutics, University of Pennsylvania, Philadelphia (D.P., O.P.B., G.A.F.).

Correspondence to Francesco Violi, MD, Institute of Clinical Medicine I, University La Sapienza, Policlinico Umberto I, 00185 Roma, Italy. E-mail violi{at}uniroma1.it

Background—Iron is an important modulator of lipid peroxidation, and its levels have been associated with the progression of atherosclerosis. Little is known about the possibility that this metal, when released from tissue stores, may modulate the reactivity of blood cell components, in particular platelets. Therefore, we investigated a possible link between iron, oxygen free radical formation, and platelet function.

Methods and Results—Human whole blood was stimulated with collagen 2 µg/mL, and an irreversible aggregation with thromboxane (Tx)B₂ formation was observed (15±4 versus 130±10 ng/mL). Deferoxamine (DSF), a specific iron chelator, and catalase, an H₂O₂ scavenger, inhibited collagen-induced whole-blood aggregation. The aggregation was accompanied by an increase in hydroxyl radical (OH·) levels (30±8 versus 205±20 nmol/L dihydroxybenzoates), which were reduced by DSF and by 2 specific OH· scavengers, mannitol and deoxyribose. Iron (Fe²⁺) dose-dependently induced platelet aggregation, TxB₂ formation (6±2 versus 135±8 ng/mL), and protein kinase C (PKC) translocation from the cytosol to the cell membrane when added to platelets that have been primed with a low concentration of collagen (0.2 µg/mL). In the same system, an increase in OH· levels was observed (37±12 versus 230±20 nmol/L dihydroxybenzoates). Mannitol and deoxyribose, but not urea, were able to reduce OH· formation, PKC activation, and platelet aggregation. Selective inhibition of PKC activity by GF 109203X prevented iron-dependent platelet aggregation without influencing OH· production.

Conclusions—The present study shows that iron can directly interact with human platelets, resulting in their activation. Its action is mediated by OH· formation and involves PKC activity. Our findings provide an additional contribution to the understanding of the mechanism(s) by which iron overload might promote atherosclerosis and coronary artery disease.

Key Words: platelets • free radicals • thromboxane • protein kinase C

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