Direct demonstration of involvement of protein kinase Cα in the Ca2+-induced platelet aggregation

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

Research output: Contribution to journalArticlepeer-review

40 Citations (Scopus)

Abstract

Platelets play critical roles in hemostasis and thrombosis through their aggregation following activation of integrin αIIbβ3. 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 αIIbβ3, 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 PKCα and β from the cytosol. The PKC-depleted cytosol lost the aggregation-supporting activity, which was recovered by the addition of purified PKCα. Furthermore, the addition of purified PKCα 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 PKCα is involved in the regulation of Ca2+-induced platelet aggregation.

Original languageEnglish
Pages (from-to)26374-26379
Number of pages6
JournalJournal of Biological Chemistry
Volume278
Issue number29
DOIs
Publication statusPublished - 2003 Jul 18

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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