Department of Neuroscience, Imaging and Clinical Science, "G. d'Annunzio" University, Chieti, Italy; CAST (Center for Advanced Studies and Technology), "G. d'Annunzio" University, Chieti, Italy.
CAST (Center for Advanced Studies and Technology), "G. d'Annunzio" University, Chieti, Italy.
Biochim Biophys Acta Mol Cell Biol Lipids. 2020 Dec;1865(12):158804. doi: 10.1016/j.bbalip.2020.158804. Epub 2020 Aug 25.
Platelet 12-lipoxygenase(p-12-LOX) is highly expressed in human platelets, and the development of p-12-LOX inhibitors has the potential to be a novel antithrombotic tool by inhibiting thrombosis without prolonging hemostasis. A chiral liquid chromatography-mass spectrometry(LC-MS/MS) method was used to assess the impact of three commercially available LOX inhibitors[esculetin(6,7-dihydroxycoumarin), ML-355(N-2-benzothiazolyl-4-[[(2-hydroxy-3-methoxyphenyl)methyl]amino]-benzenesulfonamide), CDC(cinnamyl-3,4-dihydroxy-α-cyanocinnamate) and acetylsalicylic acid(ASA; a cyclooxygenase-1 inhibitor) on the generation of prostanoids and HETEs(hydroxyeicosatetraenoic acids) in human whole blood allowed to clot for 1 h at 37 °C(serum), platelet-rich plasma(PRP) stimulated with collagen or TRAP-6(a peptide activating thrombin receptor) and washed platelets. In serum, ML-355 did not affect eicosanoid generation, while CDC caused an incomplete reduction of 12S-HETE levels; esculetin inhibited both 12S-HETE and thromboxane(TX)B production; ASA selectively affected TXB production. In washed platelets stimulated with thrombin, esculetin, and CDC inhibited both 12S-HETE and TXB while ML-355 was almost ineffective. In PRP, ML-355, CDC, and esculetin did not affect platelet aggregation associated with incomplete effects on eicosanoid biosynthesis. ASA alone or in combination with ticagrelor(a P2Y blocker) affected platelet aggregation associated with profound inhibition of TXB generation. P2Y receptor signaling contributed to platelet 12S-HETE biosynthesis in response to primary agonists. In conclusion, ML-355, esculetin, and CDC were not selective inhibitors of p-12-LOX in different cellular systems. They did not affect platelet aggregation induced in PRP by collagen or TRAP-6. The characterization of 12-LOX inhibitors on eicosanoids generated in human whole blood is useful for information on their enzyme selectivity, off-target effects, and the possible influence of plasma components on their pharmacological effects.
血小板 12-脂氧合酶（p-12-LOX）在人血小板中高度表达，开发 p-12-LOX 抑制剂有可能成为一种新型抗血栓工具，通过抑制血栓形成而不延长止血时间。采用手性液相色谱-质谱联用（LC-MS/MS）方法评估了三种市售 LOX 抑制剂[esculetin（6,7-二羟基香豆素）、ML-355（N-2-苯并噻唑基-4-[[（2-羟基-3-甲氧基苯基）甲基]氨基]苯磺酰胺）、CDC（肉桂基-3,4-二羟基-α-氰基肉桂酸）和乙酰水杨酸（ASA；环氧化酶-1 抑制剂）对人全血在 37°C 凝结 1 小时（血清）、胶原或 TRAP-6（激活凝血酶受体的肽）刺激的富血小板血浆（PRP）和洗涤血小板中前列腺素和 HETEs（羟二十碳四烯酸）生成的影响。在血清中，ML-355 不影响类花生酸的生成，而 CDC 导致 12S-HETE 水平不完全降低； esculetin 抑制 12S-HETE 和血栓素（TX）B 的产生；ASA 选择性影响 TXB 的产生。在凝血酶刺激的洗涤血小板中，esculetin 和 CDC 抑制 12S-HETE 和 TXB，而 ML-355 几乎无效。在 PRP 中，ML-355、CDC 和 esculetin 不影响与类花生酸生物合成不完全作用相关的血小板聚集。ASA 单独或与 ticagrelor（P2Y 阻断剂）联合使用会影响与 TXB 生成深度抑制相关的血小板聚集。P2Y 受体信号在对主要激动剂的反应中有助于血小板 12S-HETE 的生物合成。总之，ML-355、esculetin 和 CDC 在不同的细胞系统中不是 p-12-LOX 的选择性抑制剂。它们不影响胶原或 TRAP-6 诱导的 PRP 中血小板聚集。在人全血中生成的前列腺素的 12-LOX 抑制剂的表征对于了解其酶选择性、脱靶效应以及血浆成分对其药理作用的可能影响很有用。
