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Abstract：
ADP-induced platelet activation leads to cell surface expression of several proteins, including TF (tissue factor). The role of ADP receptors in platelet TF modulation is still unknown. We aimed to assess the (1) involvement of P2Y1 and P2Y12 receptors in ADP-induced TF exposure; (2) modulation of TFpos-platelets in anti-P2Y12-treated patients with coronary artery disease. Based on the obtained results, we revisited the intracellular localization of TF in platelets.
The effects of P2Y1 or P2Y12 antagonists on ADP-induced TF expression and activity were analyzed in vitro by flow cytometry and thrombin generation assay in blood from healthy subjects, P2Y12-/-, and patients with gray platelet syndrome. Ex vivo, P2Y12 inhibition of TF expression by clopidogrel/prasugrel/ticagrelor, assessed by VASP (vasodilator-stimulated phosphoprotein) platelet reactivity index, was investigated in coronary artery disease (n=238). Inhibition of open canalicular system externalization and electron microscopy (TEM) were used for TF localization.
In blood from healthy subjects, stimulated in vitro by ADP, the percentage of TFpos-platelets (17.3±5.5%) was significantly reduced in a concentration-dependent manner by P2Y12 inhibition only (-81.7±9.5% with 100 nM AR-C69931MX). In coronary artery disease, inhibition of P2Y12 is paralleled by reduction of ADP-induced platelet TF expression (VASP platelet reactivity index: 17.9±11%, 20.9±11.3%, 40.3±13%; TFpos-platelets: 10.5±4.8%, 9.8±5.9%, 13.6±6.3%, in prasugrel/ticagrelor/clopidogrel-treated patients, respectively). Despite this, 15% of clopidogrel good responders had a level of TFpos-platelets similar to the poor-responder group. Indeed, a stronger P2Y12 inhibition (130-fold) is required to inhibit TF than VASP. Thus, a VASP platelet reactivity index <20% (as in prasugrel/ticagrelor-treated patients) identifies patients with TFpos-platelets <20% (92% sensitivity). Finally, colchicine impaired in vitro ADP-induced TF expression but not α-granule release, suggesting that TF is open canalicular system stored as confirmed by TEM and platelet analysis of patients with gray platelet syndrome.
Data show that TF expression is regulated by P2Y12 and not P2Y1; P2Y12 antagonists downregulate the percentage of TFpos-platelets. In clopidogrel good-responder patients, assessment of TFpos-platelets highlights those with residual platelet reactivity. TF is stored in open canalicular system, and its membrane exposure upon activation is prevented by colchicine.
The effects of P2Y1 or P2Y12 antagonists on ADP-induced TF expression and activity were analyzed in vitro by flow cytometry and thrombin generation assay in blood from healthy subjects, P2Y12-/-, and patients with gray platelet syndrome. Ex vivo, P2Y12 inhibition of TF expression by clopidogrel/prasugrel/ticagrelor, assessed by VASP (vasodilator-stimulated phosphoprotein) platelet reactivity index, was investigated in coronary artery disease (n=238). Inhibition of open canalicular system externalization and electron microscopy (TEM) were used for TF localization.
In blood from healthy subjects, stimulated in vitro by ADP, the percentage of TFpos-platelets (17.3±5.5%) was significantly reduced in a concentration-dependent manner by P2Y12 inhibition only (-81.7±9.5% with 100 nM AR-C69931MX). In coronary artery disease, inhibition of P2Y12 is paralleled by reduction of ADP-induced platelet TF expression (VASP platelet reactivity index: 17.9±11%, 20.9±11.3%, 40.3±13%; TFpos-platelets: 10.5±4.8%, 9.8±5.9%, 13.6±6.3%, in prasugrel/ticagrelor/clopidogrel-treated patients, respectively). Despite this, 15% of clopidogrel good responders had a level of TFpos-platelets similar to the poor-responder group. Indeed, a stronger P2Y12 inhibition (130-fold) is required to inhibit TF than VASP. Thus, a VASP platelet reactivity index <20% (as in prasugrel/ticagrelor-treated patients) identifies patients with TFpos-platelets <20% (92% sensitivity). Finally, colchicine impaired in vitro ADP-induced TF expression but not α-granule release, suggesting that TF is open canalicular system stored as confirmed by TEM and platelet analysis of patients with gray platelet syndrome.
Data show that TF expression is regulated by P2Y12 and not P2Y1; P2Y12 antagonists downregulate the percentage of TFpos-platelets. In clopidogrel good-responder patients, assessment of TFpos-platelets highlights those with residual platelet reactivity. TF is stored in open canalicular system, and its membrane exposure upon activation is prevented by colchicine.
摘要：
背景：ADP诱导的血小板活化导致几种蛋白质的细胞表面表达，包括TF(组织因子)。ADP受体在血小板TF调节中的作用尚不清楚。我们旨在评估（1）P2Y1和P2Y12受体在ADP诱导的TF暴露中的参与；（2）在抗P2Y12治疗的冠心病患者中TFpos血小板的调节。根据获得的结果，我们重新探讨了TF在血小板中的细胞内定位。
方法：通过流式细胞术和凝血酶生成试验，体外分析了P2Y1或P2Y12拮抗剂对ADP诱导的TF表达和活性的影响。P2Y12-/-,和灰色血小板综合征患者。离体,氯吡格雷/普拉格雷/替格瑞洛P2Y12抑制TF表达，通过VASP（血管扩张剂刺激磷蛋白）血小板反应性指数评估，在冠状动脉疾病中进行了研究（n=238）。开放小管系统外化的抑制和电子显微镜（TEM）用于TF定位。
结果：在健康受试者的血液中，ADP体外刺激，TFpos-血小板的百分比(17.3±5.5%)仅通过P2Y12抑制以浓度依赖性方式显著降低(-81.7±9.5%,使用100nMAR-C69931MX)。在冠状动脉疾病中，P2Y12的抑制与ADP诱导的血小板TF表达的减少平行（VASP血小板反应性指数：17.9±11％，20.9±11.3%，40.3±13%；TFpos-血小板：10.5±4.8%，9.8±5.9％，13.6±6.3%,普拉格雷/替格瑞洛/氯吡格雷治疗的患者，分别)。尽管如此,15％的氯吡格雷良好反应者的TFpos-血小板水平与不良反应者相似。的确,与VASP相比,需要更强的P2Y12抑制(130倍)来抑制TF。因此，VASP血小板反应性指数<20%(如普拉格雷/替格瑞洛治疗的患者)确定TFpos-血小板<20%的患者(92%敏感性).最后,秋水仙碱损害体外ADP诱导的TF表达，但不损害α-颗粒释放，提示TF是开放的小管系统储存，如TEM和灰色血小板综合征患者的血小板分析所证实。
结论：数据显示TF表达受P2Y12而非P2Y1调节；P2Y12拮抗剂下调TFpos-血小板的百分比。在氯吡格雷反应良好的患者中，对TFpos血小板的评估突出了那些具有残余血小板反应性的血小板。TF存储在开放的小管系统中，秋水仙碱可以防止其激活后的膜暴露。
方法：通过流式细胞术和凝血酶生成试验，体外分析了P2Y1或P2Y12拮抗剂对ADP诱导的TF表达和活性的影响。P2Y12-/-,和灰色血小板综合征患者。离体,氯吡格雷/普拉格雷/替格瑞洛P2Y12抑制TF表达，通过VASP（血管扩张剂刺激磷蛋白）血小板反应性指数评估，在冠状动脉疾病中进行了研究（n=238）。开放小管系统外化的抑制和电子显微镜（TEM）用于TF定位。
结果：在健康受试者的血液中，ADP体外刺激，TFpos-血小板的百分比(17.3±5.5%)仅通过P2Y12抑制以浓度依赖性方式显著降低(-81.7±9.5%,使用100nMAR-C69931MX)。在冠状动脉疾病中，P2Y12的抑制与ADP诱导的血小板TF表达的减少平行（VASP血小板反应性指数：17.9±11％，20.9±11.3%，40.3±13%；TFpos-血小板：10.5±4.8%，9.8±5.9％，13.6±6.3%,普拉格雷/替格瑞洛/氯吡格雷治疗的患者，分别)。尽管如此,15％的氯吡格雷良好反应者的TFpos-血小板水平与不良反应者相似。的确,与VASP相比,需要更强的P2Y12抑制(130倍)来抑制TF。因此，VASP血小板反应性指数<20%(如普拉格雷/替格瑞洛治疗的患者)确定TFpos-血小板<20%的患者(92%敏感性).最后,秋水仙碱损害体外ADP诱导的TF表达，但不损害α-颗粒释放，提示TF是开放的小管系统储存，如TEM和灰色血小板综合征患者的血小板分析所证实。
结论：数据显示TF表达受P2Y12而非P2Y1调节；P2Y12拮抗剂下调TFpos-血小板的百分比。在氯吡格雷反应良好的患者中，对TFpos血小板的评估突出了那些具有残余血小板反应性的血小板。TF存储在开放的小管系统中，秋水仙碱可以防止其激活后的膜暴露。
