Centro Cardiologico Monzino IRCCS, Milan, Italy (M.B., A. Becchetti, N.C., M. Conti, P.C., A. Bonomi, D.T., P.J.W., J.C., G.M., M. Camera).
Department of Pharmaceutical Sciences (G.E.R., C.P., M. Camera), Università degli Studi di Milano, Italy.
Arterioscler Thromb Vasc Biol. 2023 Oct;43(10):2042-2057. doi: 10.1161/ATVBAHA.123.319099. Epub 2023 Aug 17.
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 P2Y and P2Y receptors in ADP-induced TF exposure; (2) modulation of TF-platelets in anti-P2Y-treated patients with coronary artery disease. Based on the obtained results, we revisited the intracellular localization of TF in platelets.
The effects of P2Y or P2Y antagonists on ADP-induced TF expression and activity were analyzed in vitro by flow cytometry and thrombin generation assay in blood from healthy subjects, P2Y, and patients with gray platelet syndrome. Ex vivo, P2Y 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 TF-platelets (17.3±5.5%) was significantly reduced in a concentration-dependent manner by P2Y inhibition only (-81.7±9.5% with 100 nM AR-C69931MX). In coronary artery disease, inhibition of P2Y is paralleled by reduction of ADP-induced platelet TF expression (VASP platelet reactivity index: 17.9±11%, 20.9±11.3%, 40.3±13%; TF-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 TF-platelets similar to the poor-responder group. Indeed, a stronger P2Y 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 TF-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 P2Y and not P2Y; P2Y antagonists downregulate the percentage of TF-platelets. In clopidogrel good-responder patients, assessment of TF-platelets highlights those with residual platelet reactivity. TF is stored in open canalicular system, and its membrane exposure upon activation is prevented by colchicine.
ADP 诱导的血小板活化导致细胞表面表达几种蛋白质，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)P2Y 和 P2Y 受体在 ADP 诱导的 TF 暴露中的作用；(2)在接受抗 P2Y 治疗的冠心病患者中，TF-血小板的调节。根据获得的结果，我们重新研究了 TF 在血小板中的细胞内定位。
通过流式细胞术和血栓生成试验，分析了体外 P2Y 或 P2Y 拮抗剂对 ADP 诱导的 TF 表达和活性的影响，该试验在健康受试者、P2Y 和患有灰色血小板综合征的患者的血液中进行。在体外，通过 VASP（血管扩张刺激磷酸蛋白）血小板反应性指数评估氯吡格雷/普拉格雷/替格瑞洛对 TF 表达的 P2Y 抑制作用，对 238 例冠心病患者进行了研究。使用开放小管系统外化和电子显微镜 (TEM) 抑制 TF 定位。
在健康受试者的血液中，体外用 ADP 刺激后，TF-血小板的百分比（17.3±5.5%）通过 P2Y 抑制以浓度依赖性方式显著降低（100 nM AR-C69931MX 时为-81.7±9.5%）。在冠心病中，P2Y 抑制与 ADP 诱导的血小板 TF 表达减少平行（VASP 血小板反应性指数：分别在接受普拉格雷/替格瑞洛/氯吡格雷治疗的患者中为 17.9±11%、20.9±11.3%、40.3±13%；TF-血小板：10.5±4.8%、9.8±5.9%、13.6±6.3%）。尽管如此，15%的氯吡格雷反应良好的患者 TF-血小板水平与反应不良的患者相似。事实上，需要更强的 P2Y 抑制（130 倍）来抑制 TF，而不是 VASP。因此，VASP 血小板反应性指数<20%（如在接受普拉格雷/替格瑞洛治疗的患者中）可识别 TF-血小板<20%（92%敏感性）的患者。最后，秋水仙碱可抑制体外 ADP 诱导的 TF 表达，但不抑制α-颗粒释放，表明 TF 作为在 TEM 和患有灰色血小板综合征的患者的血小板分析中得到证实的开放小管系统储存。
数据表明，TF 表达受 P2Y 调节，不受 P2Y 调节；P2Y 拮抗剂下调 TF-血小板的百分比。在氯吡格雷反应良好的患者中，评估 TF-血小板突出了那些具有残留血小板反应性的患者。TF 储存在开放小管系统中，秋水仙碱可防止其在激活时暴露于细胞膜。
