Published Online
on April 29, 2002

A more recent version of this article appeared on May 28, 2002

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Submitted on December 12, 2001
Revised on March 8, 2002
Accepted on March 10, 2002

Functional Significance of Adenosine 5'-Diphosphate Receptor (P2Y₁₂) in Platelet Activation Initiated by Binding of von Willebrand Factor to Platelet GP Ib Induced by Conditions of High Shear Rate

Shinya Goto MD^*, Noriko Tamura BS, Koji Eto MD, Yasuo Ikeda MD, and Shunnosuke Handa MD

From the Division of Cardiology (S.G., N.T., K.E., S.H.), Department of Medicine, Tokai University School of Medicine, Kanagawa, Japan; and the Department of Medicine (Y.I.), Keio University School of Medicine, Tokyo, Japan.

^* To whom correspondence should be addressed. E-mail: shinichi{at}is.icc.u-tokai.ac.jp.

Background—The role of the adenosine 5`-diphosphate receptor P2Y₁₂ in platelet activation initiated by the von Willebrand factor (VWF)--GP Ib interaction under high shear rate was investigated.

Methods and Results—Blood samples were obtained from 11 donors. Shear-induced platelet aggregation was detected by optically modified cone-plate viscometer. Shear-induced VWF binding, P-selectin expression, and microparticle release were detected by flow cytometry. Platelet interaction with immobilized VWF was also investigated by parallel-plate flow chamber equipped with epifluorescent videomicroscopy. Effects of a selective P2Y₁₂ antagonist AR-C69931 MX were tested. AR-C69931 MX inhibited shear-induced platelet aggregation in a dose-dependent manner, achieving the maximum inhibition at 100 nmol/L. The extent of aggregation after exposure to a shear rate of 10 800 s^-1 for 6 minutes in the presence of 100 nmol/L AR-C69931 MX was 32.4±8.2% (mean±SD), which was significantly lower than the value in the controls of 69.7±9.6% (P<0.01). The inhibiting effects of AR-C69931 MX were reversed by exogenous addition of adenosine 5`-diphosphate. Shear-induced VWF binding and P-selectin surface translocation, which occurred in 4696±911 and 5964±784, respectively, of 10 000 measured platelets, was also inhibited by AR-C69931 MX (100 nmol/L) to 1948±528 and 2797±718, respectively (P=0.0018 and P=0.0009). Microparticle release was similarly inhibited. In a flow chamber experiment, firm platelet attachment on immobilized VWF was inhibited by AR-C69931 MX, whereas transient interaction was not influenced. All the above reactions were completely inhibited by blocking VWF--GP Ib interaction.

Conclusions—We have demonstrated that the stimulation of P2Y₁₂ is involved in platelet activation initiated by the binding of VWF to GP Ib induced by a high shear rate.

Key words: platelets • thrombosis • glycoproteins • von Willebrand factor • receptors

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