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Critical role of ADP interaction with P2Y 12 receptor in the maintenance of α IIb β 3 activation: association with Rap1B activation
Author(s) -
KAMAE T.,
SHIRAGA M.,
KASHIWAGI H.,
KATO H.,
TADOKORO S.,
KURATA Y.,
TOMIYAMA Y.,
KANAKURA Y.
Publication year - 2006
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2006.01941.x
Subject(s) - platelet activation , platelet , chemistry , thrombin , protein kinase c , p2y12 , adenosine diphosphate , microbiology and biotechnology , biophysics , biochemistry , kinase , medicine , biology , platelet aggregation
Summary. Objective: Platelet integrin α IIb β 3 plays a crucial role in platelet aggregation, and the affinity of α IIb β 3 for fibrinogen is dynamically regulated. Employing modified ligand‐binding assays, we analyzed the mechanism by which α IIb β 3 maintains its high‐affinity state. Methods and results: Washed platelets adjusted to 50 × 10 3 μ L −1 were stimulated with 0.2 U mL −1 thrombin or 5 μ m U46619 under static conditions. After the completion of α IIb β 3 activation and granule secretion, different kinds of antagonists were added to the activated platelets. The activated α IIb β 3 was then detected by fluorescein isothiocyanate (FITC)‐labeled PAC1. The addition of 1 μ m AR‐C69931MX (a P2Y 12 antagonist) or 1 m m A3P5P (a P2Y 1 antagonist) disrupted the sustained α IIb β 3 activation by ∼92% and ∼38%, respectively, without inhibiting CD62P or CD63 expression. Dilution of the platelet preparation to 500 μ L −1 also disrupted the sustained α IIb β 3 activation, and the disruption by such dilution was abrogated by the addition of exogenous adenosine 5′‐diphosphate (ADP) in a dose‐dependent fashion. The amounts of ADP released from activated platelets determined by high‐performance liquid chromatography were compatible with the amounts of exogenous ADP required for the restoration. We next examined the effects of antagonists on protein kinase C (PKC) and Rap1B activation induced by 0.2 U mL −1 thrombin. Thrombin induced long‐lasting PKC and Rap1B activation. AR‐C69931MX markedly inhibited Rap1B activation without inhibiting PKC activation. Conclusions: Our data indicate that the continuous interaction between released ADP and P2Y 12 is critical for the maintenance of α IIb β 3 activation.