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J. Biol. Chem., Vol. 278, Issue 29, 26374-26379, July 18, 2003

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Direct Demonstration of Involvement of Protein Kinase C in the Ca²⁺-induced Platelet Aggregation^*

Arata Tabuchi, Akira Yoshioka , Tomohito Higashi, Ryutaro Shirakawa, Hiroaki Nishioka , Toru Kita ¶ and Hisanori Horiuchi ||

From the Department of Geriatric Medicine and ^¶Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, 606-8507 Kyoto, Japan

Platelets play critical roles in hemostasis and thrombosis through their aggregation following activation of integrin _IIb3. However, the molecular mechanism of the integrin activation inside platelets remains largely unknown. Pharmacological experiments have demonstrated that protein kinase C (PKC) plays an important role in platelet aggregation. Because PKC inhibitors can have multiple substrates and given that non-PKC-phorbol ester-binding signaling molecules have been demonstrated to play important roles, the precise involvement of PKC in cellular functions requires re-evaluation. Here, we have established an assay for analyzing the Ca²⁺-induced aggregation of permeabilized platelets. The aggregation of platelets was inhibited by the addition of the arginine-glycine-aspartate-serine peptide, an integrin-binding peptide inhibitor of _IIb3, suggesting that the aggregation was mediated by the integrin. The aggregation was also dependent on exogenous ATP and platelet cytosol, indicating the existence of essential cytosolic factors required for the aggregation. To examine the role of PKC in the aggregation assay, we immunodepleted PKC and from the cytosol. The PKC-depleted cytosol lost the aggregation-supporting activity, which was recovered by the addition of purified PKC. Furthermore, the addition of purified PKC in the absence of cytosol did not support the aggregation, whereas the cytosol containing less PKC supported it efficiently, suggesting that additional factors besides PKC would also be required. Thus, we directly demonstrated that PKC is involved in the regulation of Ca²⁺-induced platelet aggregation.

Received for publication, December 5, 2002 , and in revised form, March 31, 2003.

^* This work was supported by Research Grants from Ministry of Education, Science, Sports, and Culture of Japan (Grants 15590740 (to H. H.) and 12CE2006 and 13307034 (to T. K.)), Research Grants from Ministry of Health, Labor, and Welfare of Japan (Comprehensive Research on Aging and Health Grant H14-choju-012 (to T. K. and H. H.)), and partially by grants from Takeda Science Foundation, Suzuken Memorial Foundation, Study Group of Molecular Cardiology and Novartis Foundation for Gerontrogical Research (to H. H.). 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.

Present address: Dept. of Internal Medicine, Mitsubishi Kyoto Hospital, 615-8087 Kyoto, Japan.

Present address: Sir William Dunn School of Pathology, University of Oxford, South Parks Rd., Oxford OX1 3RE, United Kingdom.

^|| To whom correspondence should be addressed. E-mail: horiuchi{at}kuhp.kyoto-u.ac.jp.

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