Direct demonstration of involvement of protein kinase Calpha in the Ca2+-induced platelet aggregation.
Platelets play critical roles in hemostasis and thrombosis through their aggregation following activation of integrin alphaIIbbeta3. 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 alphaIIbbeta3, 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 PKCalpha and beta from the cytosol. The PKC-depleted cytosol lost the aggregation-supporting activity, which was recovered by the addition of purified PKCalpha. Furthermore, the addition of purified PKCalpha 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 PKCalpha is involved in the regulation of Ca2+-induced platelet aggregation.
Department of Geriatric Medicine and Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, 606-8507 Kyoto, Japan.
Arata
A
Tabuchi
Akira
A
Yoshioka
Tomohito
T
Higashi
Ryutaro
R
Shirakawa
Hiroaki
H
Nishioka
Toru
T
Kita
Hisanori
H
Horiuchi
D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology*; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D066298:In Vitro Techniques; D009842:Oligopeptides / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects* / Q000502:physiology*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000037:antagonists & inhibitors; D011493:Protein Kinase C / Q000097:blood / Q000494:pharmacology / Q000502:physiology*; D051571:Protein Kinase C-alpha
D009842:Oligopeptides; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; C046617:arginyl-glycyl-aspartyl-serine; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D002118:Calcium