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J. Biol. Chem., Vol. 283, Issue 36, 24460-24468, September 5, 2008

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Adenylic Dinucleotides Produced by CD38 Are Negative Endogenous Modulators of Platelet Aggregation^*

Mirko Magnone, Giovanna Basile, Debora Bruzzese, Lucrezia Guida, Maria Grazia Signorello, Madhu Parakkottil Chothi, Santina Bruzzone, Enrico Millo, Ai-Dong Qi^¶, Robert A. Nicholas^¶, Matthias U. Kassack^||, Giuliana Leoncini, and Elena Zocchi¹

From the Department of Experimental Medicine, Section of Biochemistry and Center of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV, 1 16132 Genova, Italy, the Advanced Biotechnology Center, Largo Rosanna Benzi 10, 16132 Genova, Italy, the ^¶Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599-7365, and the ^||Institute of Pharmaceutical and Medicinal Chemistry, University of Duesseldorf, Universitaetsstrasse 1, D-40225 Duesseldorf, Germany

Diadenosine 5',5'''-P1,P2-diphosphate (Ap2A) is one of the adenylic dinucleotides stored in platelet granules. Along with proaggregant ADP, it is released upon platelet activation and is known to stimulate myocyte proliferation. We have previously demonstrated synthesis of Ap2A and of two isomers thereof, called P18 and P24, from their high pressure liquid chromatography retention time, by the ADP-ribosyl cyclase CD38 in mammalian cells. Here we show that Ap2A and its isomers are present in resting human platelets and are released during thrombin-induced platelet activation. The three adenylic dinucleotides were identified by high pressure liquid chromatography through a comparison with the retention times and the absorption spectra of purified standards. Ap2A, P18, and P24 had no direct effect on platelet aggregation, but they inhibited platelet aggregation induced by physiological agonists (thrombin, ADP, and collagen), with mean IC₅₀ values ranging between 5 and 15 µM. Moreover, the three dinucleotides did not modify the intracellular calcium concentration in resting platelets, whereas they significantly reduced the thrombin-induced intracellular calcium increase. Through binding to the purinergic receptor P2Y₁₁, exogenously applied Ap2A, P18, and P24 increased the intracellular cAMP concentration and stimulated platelet production of nitric oxide, the most important endogenous antiaggregant. The presence of Ap2A, P18, and P24 in resting platelets and their release during thrombin-induced platelet activation at concentrations equal to or higher than the respective IC₅₀ value on platelet aggregation suggest a role of these dinucleotides as endogenous negative modulators of aggregation.

Received for publication, December 28, 2007 , and in revised form, July 3, 2008.

^* This work was supported in part by grants from the Associazione Italiana per la Ricerca sul Cancro; from the Italian Ministry of Education, University, and Scientific Research (MIUR-PRIN 2003, MIUR FIRB RBAUO19A3C, MIUR FIRB RBNE01ERXR and MIUR FIRB RBLA039LSF); and from the University of Genova and Fondazione Cassa di Risparmio di Genova e Imperia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV, 1 16132 Genova, Italy. Tel.: 0103538158; Fax: 010354415; E-mail: ezocchi{at}unige.it.

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