Critical role of ADP interaction with P2Y12 receptor in the maintenance of alpha(IIb)beta3 activation: association with Rap1B activation.
OBJECTIVE
Platelet integrin alpha(IIb)beta3 plays a crucial role in platelet aggregation, and the affinity of alpha(IIb)beta3 for fibrinogen is dynamically regulated. Employing modified ligand-binding assays, we analyzed the mechanism by which alpha(IIb)beta3 maintains its high-affinity state.


RESULTS
Washed platelets adjusted to 50 x 10(3) microL(-1) were stimulated with 0.2 U mL(-1) thrombin or 5 microm U46619 under static conditions. After the completion of alpha(IIb)beta3 activation and granule secretion, different kinds of antagonists were added to the activated platelets. The activated alpha(IIb)beta3 was then detected by fluorescein isothiocyanate (FITC)-labeled PAC1. The addition of 1 mum AR-C69931MX (a P2Y12 antagonist) or 1 mm A3P5P (a P2Y1 antagonist) disrupted the sustained alpha(IIb)beta3 activation by approximately 92% and approximately 38%, respectively, without inhibiting CD62P or CD63 expression. Dilution of the platelet preparation to 500 microL(-1) also disrupted the sustained alpha(IIb)beta3 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 P2Y12 is critical for the maintenance of alpha(IIb)beta3 activation.
Kamae
T
T
Department of Hematology and Oncology, Graduate School of Medicine C9, Osaka University, Suita, Osaka, Japan.
Shiraga
M
M
Kashiwagi
H
H
Kato
H
H
Tadokoro
S
S
Kurata
Y
Y
Tomiyama
Y
Y
Kanakura
Y
Y
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000378:metabolism* / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D015539:Platelet Activation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2 / Q000172:deficiency / Q000276:immunology / Q000378:metabolism*; D058925:Receptors, Purinergic P2Y12; D015398:Signal Transduction; D013917:Thrombin / Q000494:pharmacology; D020668:rap GTP-Binding Proteins / Q000378:metabolism*
D000911:Antibodies, Monoclonal; C548576:P2RY12 protein, human; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058925:Receptors, Purinergic P2Y12; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; C117446:cangrelor; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D011493:Protein Kinase C; D013917:Thrombin; C116753:RAP1B protein, human; D020668:rap GTP-Binding Proteins