氯吡格雷抑制冠状动脉粥样硬化性心脏病患者血小板受体活化的特征

Activating profile of platelet receptors in patients with coronary disease after clopidogrel intake

目的 探讨冠状动脉粥样硬化性心脏病患者服用氯吡格雷后血小板受体抑制与总活性短期改变的特征.方法 选择冠状动脉粥样硬化性心脏病患者32例,在服用100 mg/d阿司匹林的同时,分别在服用氯吡格雷前、300 mg负荷剂量后10 h和36 h（维持剂量75 mg）分3次采血检测.用AA和ADP作为诱导剂测定血小板聚集率,使用血栓弹力图仪配套试剂盒中的高岭土（Kaolin）试剂、AA、ADP和ActivatorF（（R）） 检测并计算血小板INH;在FACSCalibur流式细胞仪上检测血小板活化标志物CD62p和VASP磷酸化水平（以PRI表示）,分别了解血小板分泌活性和P2Y12受体被抑制的情况.比较各指标在服药前后的动态变化趋势,并评价其在反映血小板受体活化功能水平的价值.结果 ADP诱导的血小板INH在服药前为（11.5±9.3）%,服药后10 h升至（42.5±29.1）%,与服药前比较差异有统计学意义（t=3.155,P〈0.05）,服药后36 h降至（20.4±13.1）%,与服药前比较差异无统计学意义（t=2.078,P〉0.05）;由AA诱导的血小板INH服药前为（56.6±36.6）%,服药后10 h升至（83.0±27.3）%,与服药前比较差异无统计学意义（t=2.086,P〉0.05）,服药后36 h升至（85.4±20.8）%,与服药前比较差异无统计学意义（t=1.888,P〉0.05）;表明300 mg负荷剂量氯吡格雷对ADP诱导作用的抑制在36 h内未能稳定.由ADP诱导的血小板聚集率在服药前为（53.7±14.1）%,服药后10 h降至（49.2＋22.8）%,与服药前比较差异无统计学意义（t=0.656,P〉0.05）,服药后36 h降至（40.7±12.8）%,与服药前比较差异有统计学意义（t=2.418,P〈0.05）;由AA诱导的血小板聚集率在服药前为（34.3±18.1）%,服药后10 h降至（17.4±13.1）%,与服药前比较差异有统计学意义（t=3.134,P〈0.05）,服药后36 h降至（14.6±5.1）%,与服药前比较差异有统计学意义（t=2.532,P〈0.05）.PRI在服药前为（78.6±22.3）%,服药后10 h降至（70.7±9.4）%,与服药前比较差异无统计学意义（t=1.194,P〉0.05）,服药后36 h降至（59.6±28.0）%,与服药前比较差异有统计学意义（t=1.930,P〈0.05）,说明氯吡格雷在36 h内尚未对ADP受体形成有力的抑制效果.CD62p在服药前为（7.5±1.4）%,服药后10 h降至（4.2±1.1）%,与服药前比较差异有统计学意义（t=18.027,P〈0.05）,服药后36 h降至（4.3±0.2）%,与服药前比较差异无统计学意义（t=2.908,P〉0.05）.CD62p反映的分泌活性所受抑制比较明显.相比而言,环氧化酶-1途径所受的抑制更为显著,而P2Y12受体所受抑制却因方法差异而不尽相同.结论 从诱导的血小板活化在氯吡格雷和阿司匹林联合抑制下显著减低,ADP受体直到服用负荷剂量氯吡格雷后36 h才出现显著抑制.全血中血小板功能反映了血小板与其他成分交互作用下的总活性,10 h内效果不明显.

Objective To study the characteristic of inhibition on platelet P2Y12 and short-term change after clopidogrel intake in patients with cardiovascular disease. Methods Thirty-two patients with cardiovascular disease were enrolled. Samples at baseline, 10 h and 36 h after 300 mg loading dose and 75 mg/d maintenance dose of clopidogrel with 100 mg/d Aspirin intake were measured respectively. Platelet aggregation （PAgT） was measured on thromboelastograph（TEG） induced by ADP/AA. INH was detected and calculated activated by Kaolin, AA, ADP and Activator（（R）） in the TEG reagent. CD62p and VASP phosphorylation （PRI）, platelet activation markers were tested with FACSCalibur Flow Cytometry, and platelet secretion activity and suppression of P2Y12 receptor were detected respectively. The changes of indicators were compared before and after clopidogrel intake, and evaluate their function in platelet receptor activation. Results INHADP at baseline was （11.5 ±9.3）%, and increased to （42.5 ±29.1）% statistically （t =3.155, P〈0.05） after taking the P2Y12 at 10 h, but decreased to （20.4±13.1）% at 36 h, non-statistical to baseline （t = 2.078, P 〉 0. 05） , INHAA increases from baseline level （56. 6 ± 36. 6） % to （83.0 ±27. 3）% at 10h（t=2.086,P〉0.05） and （85. 4 ±20. 8）% at 36 h （t= 1. 888, P〉0.05）, no statistical defferences were found. Inhibition on platelet activativation induced by ADP function well till 36 h after 300 mg loading dose. PAgTADP decrease from （53. 7 ± 14. 1）% at baseline to （49. 2 ±22. 8）% at 10 h non-statistically （t=0.656, P〉0.05）, and （40.7±12.8）% at 36 h statistically （t=2.418, P〈0.05）, however PAgTAA decrease at both 10 h and 36 h statiscally, from （34. 3 ± 18. 1） % to （17.4 ± 13. 1） % , （t=3.134, P〈0.05） and （14.6±5.1）%, （t=2.532, P〈0.05）, respectively. Data of PAgT was not corresponding to that from TEG for the difference in sample type partly. PRI in VASP assay was （78. 6 ± 22.3）% before loading dose, and decreased to （70.7 ±9.4）% at 10 h without significance （t = l. 194, P〉0.05） and （59.6 ±28.0）% at 36 h （t=1.930,P〈0.05） statistically, similarly to INHADP,indicating that within 36 h clopidogrel did not have strong inhibitory effect on the ADP receptor. On the contrary, CD62p changed from （7. 5 ± 1. 4） % at baseline to （4. 2 ± 1. 1） % statistically （10 h, t = 18. 027, P 〈 0. 05） and （ 4. 3 ± 0. 2 ） % non-statistically （36 h, t = 2. 908, P 〉 0. 05 ）. Inhibition of secretion activity reflected by CD62p was significant. In contrast, it was more obvious inhibition in COX-1 passway, while the inhibition of P2Y12 receptor varied due to assay difference. Conclusions AA-induced platelet activation is significantly decreased in the inhibition of clopidogrel and aspirin, while ADP receptor is significantly inhibited until 36 h after the loading dose of clopidogrel. Platelet function in whole blood reflects total activity of platelet interaction with other components, in which no significant inhibition could be witnessed within 10 h.