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J. Biol. Chem., Vol. 279, Issue 25, 26266-26273, June 18, 2004

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ATP Augments von Willebrand Factor-dependent Shear-induced Platelet Aggregation through Ca²⁺-Calmodulin and Myosin Light Chain Kinase Activation^*

Cécile Oury, Elsie Sticker, Heidi Cornelissen, Rita De Vos¶, Jos Vermylen, and Marc F. Hoylaerts||

From the Center for Molecular and Vascular Biology and the ^¶Laboratory of Morphology and Molecular Pathology, University of Leuven, 3000 Leuven, Belgium

Shear stress triggers von Willebrand factor (VWF) binding to platelet glycoprotein Ib and subsequent integrin _IIb3-dependent platelet aggregation. Concomitantly, nucleotides are released from plateletdense granules, and ADP is known to contribute to shear-induced platelet aggregation (SIPA). We found that the impaired SIPA of platelets from a Hermansky-Pudlak patient lacking dense granules was restored by exogenous L-,-methylene ATP, a stable P2X₁ agonist, as well as by ADP, confirming that in addition to ADP (via P2Y₁ and P2Y₁₂), ATP (via P2X₁) also contributes to SIPA. Likewise, SIPA of apyrase-treated platelets was restored upon P2X₁ activation with L-,-methylene ATP, which promoted granule centralization within platelets and stimulated P-selectin expression, which is a marker of -granule release. In addition, during SIPA, platelet degranulation required both extracellular Ca²⁺ and VWF-glycoprotein Ib interactions without involving _IIb3. Neither platelet release nor SIPA was affected by protein kinase C inactivation, even though protein kinase C blockade inhibits platelet responses to collagen and thrombin in stirring conditions. In contrast, inhibiting myosin light chain (MLC) kinase with ML-7 reduced platelet release and SIPA by 30%. Accordingly, the potentiating effect of P2X₁ stimulation on the aggregation of apyrase-treated platelets coincided with intensified phosphorylation of MLC and was abrogated by ML-7. SIPA-induced MLC phosphorylation occurred exclusively through released nucleotides and selective antagonism of P2X₁ with MRS2159-reduced SIPA, ATP release, and potently inhibited MLC phosphorylation. We conclude that the P2X₁ ion channel induces MLC-mediated cytoskeletal rearrangements, thus contributing to SIPA and degranulation during VWF-triggered platelet activation.

Received for publication, February 24, 2004 , and in revised form, April 14, 2004.

^* This work was supported by Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (FWO) Project G.0227.03 and by Geconcerteerde Onderzoeksactie/2004/09. 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.

Recipient of a postdoctoral research mandate from the FWO.

^|| To whom correspondence should be addressed: University of Leuven, Center for Molecular and Vascular Biology, Herestraat 49, 3000 Leuven, Belgium. Tel.: 32-16-346145; Fax: 32-16-345990; E-mail: marc.hoylaerts{at}med.kuleuven.ac.be.

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