Kamae T, Shiraga M, Kashiwagi H, Kato H, Tadokoro S, Kurata Y, Tomiyama Y, Kanakura Y
Department of Hematology and Oncology, Graduate School of Medicine C9, Osaka University, Suita, Osaka, Japan.
J Thromb Haemost. 2006 Jun;4(6):1379-87. doi: 10.1111/j.1538-7836.2006.01941.x.
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.
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.
Our data indicate that the continuous interaction between released ADP and P2Y12 is critical for the maintenance of alpha(IIb)beta3 activation.
血小板整合素α(IIb)β3在血小板聚集中起关键作用，且α(IIb)β3对纤维蛋白原的亲和力受到动态调节。我们采用改良的配体结合试验，分析了α(IIb)β3维持其高亲和力状态的机制。
将洗涤后的血小板调整至50×10³μL⁻¹，在静态条件下用0.2 U/mL凝血酶或5 μM U46619刺激。在α(IIb)β3激活和颗粒分泌完成后，向活化的血小板中加入不同种类的拮抗剂。然后用异硫氰酸荧光素（FITC）标记的PAC1检测活化的α(IIb)β3。加入1 μM AR-C69931MX（一种P2Y12拮抗剂）或1 mM A3P5P（一种P2Y1拮抗剂）分别使α(IIb)β3的持续活化破坏约92%和约38%，且不抑制CD62P或CD63的表达。将血小板制剂稀释至500 μL⁻¹也会破坏α(IIb)β3的持续活化，而这种稀释引起的破坏可通过以剂量依赖方式添加外源性腺苷5'-二磷酸（ADP）而消除。通过高效液相色谱法测定活化血小板释放的ADP量与恢复所需的外源性ADP量相符。接下来，我们研究了拮抗剂对0.2 U/mL凝血酶诱导的蛋白激酶C（PKC）和Rap1B活化的影响。凝血酶诱导PKC和Rap1B的持久活化。AR-C69931MX显著抑制Rap1B活化而不抑制PKC活化。
我们的数据表明，释放的ADP与P2Y12之间的持续相互作用对于维持α(IIb)β3活化至关重要。
