Department of Experimental Medicine, Sapienza University of Rome, Italy.
Julius-Maximilians-Universität Würzburg, Würzburg, Germany.
Thromb Haemost. 2018 Mar;118(3):490-501. doi: 10.1055/s-0038-1629920. Epub 2018 Feb 15.
Platelet multidrug resistance protein 4 (MRP4) plays a modulating role on platelet activation. Platelet function and thrombus formation are impaired in MRP4 knockout mice models, and, among aspirin-treated patients, high on-aspirin residual platelet reactivity (HARPR) positively correlates with MRP4 levels. To better understand the effects of MRP4 on platelet function, the aim of this investigation was to assess the impact of cilostazol-induced inhibition of MRP4-mediated transport and assess aspirin-induced antiplatelet effects and rates of HARPR in human subjects.Cilostazol-dependent inhibition of MRP4-mediated transport was assessed with the release of the fluorescent adduct bimane-glutathione and aspirin entrapment. Effect of Cilostazol on cAMP inhibition was evaluated by vasodilator-stimulated phosphoprotein (VASP). Platelet function was studied by collagen and TRAP-6-induced platelet aggregation and secretion.Cilostazol reduced the release of bimane-glutathione and enhanced aspirin entrapment demonstrating an inhibitory effect on MRP4 in platelets. VASP phosphorylation was absent until 10 seconds after addition of cilostazol, and becomes evident after 30 seconds. An inhibitory effect on platelet aggregation and secretion was found in activated platelets, with threshold concentration of agonists, 10 seconds after addition of cilostazol, supporting a role of MRP4 on platelet function that is cAMP independent. Cilostazol effects were also shown in aspirin-treated platelets. A reduction of platelet aggregation and secretion were observed in aspirin-treated patients with HARPR.This study supports the role of MRP4 on modulating platelet function which occurs through cAMP-independent mechanisms. Moreover, inhibition of MRP4 induced by cilostazol enhances aspirin-induced antiplatelet effects and reduces HARPR.
多药耐药相关蛋白 4（MRP4）在血小板激活中起调节作用。在 MRP4 基因敲除小鼠模型中，血小板功能和血栓形成受损，在接受阿司匹林治疗的患者中，高阿司匹林残留血小板反应性（HARPR）与 MRP4 水平呈正相关。为了更好地了解 MRP4 对血小板功能的影响，本研究旨在评估西洛他唑抑制 MRP4 介导的转运对血小板功能的影响，并评估西洛他唑对人血小板中阿司匹林的抗血小板作用和 HARPR 发生率的影响。用荧光加合物双马来酰亚胺-谷胱甘肽的释放和阿司匹林的捕获来评估西洛他唑对 MRP4 介导的转运的抑制作用。用环磷酸腺苷（cAMP）抑制的血管扩张刺激磷蛋白（VASP）来评估西洛他唑的作用。通过胶原蛋白和 TRAP-6 诱导的血小板聚集和分泌来研究血小板功能。西洛他唑减少双马来酰亚胺-谷胱甘肽的释放并增强阿司匹林的捕获，表明对血小板中的 MRP4 具有抑制作用。直到加入西洛他唑 10 秒后，VASP 磷酸化才消失，30 秒后才变得明显。在激活的血小板中发现对聚集和分泌的抑制作用，激动剂的阈值浓度在加入西洛他唑 10 秒后，这表明 MRP4 对血小板功能具有 cAMP 独立的作用。西洛他唑的作用也在阿司匹林治疗的血小板中显示出来。在 HARPR 的阿司匹林治疗患者中观察到血小板聚集和分泌减少。本研究支持 MRP4 调节血小板功能的作用，这种作用通过 cAMP 独立的机制发生。此外，西洛他唑抑制 MRP4 可增强阿司匹林的抗血小板作用并降低 HARPR。
