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Pharmacological characterization of the biosynthesis of prostanoids and hydroxyeicosatetraenoic acids in human whole blood and platelets by targeted chiral lipidomics analysis.
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids ( IF 4.8 ) Pub Date : 2020-08-25 , DOI: 10.1016/j.bbalip.2020.158804
Stefania Tacconelli 1 , Rosa Fullone 1 , Melania Dovizio 1 , Graziana Pizzicoli 1 , Stephanie Marschler 1 , Annalisa Bruno 1 , Mirco Zucchelli 2 , Annalisa Contursi 1 , Patrizia Ballerini 1 , Paola Patrignani 1
Affiliation  

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)B2 production; ASA selectively affected TXB2 production. In washed platelets stimulated with thrombin, esculetin, and CDC inhibited both 12S-HETE and TXB2 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 P2Y12 blocker) affected platelet aggregation associated with profound inhibition of TXB2 generation. P2Y12 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.



中文翻译:

靶向手性脂质组学分析在人全血和血小板中前列腺素和羟基二十碳四烯酸生物合成的药理学表征。

血小板12-脂氧合酶(p-12-LOX)在人体血小板中高度表达,p-12-LOX抑制剂的发展具有通过抑制血栓形成而不延长止血的潜力成为一种新型的抗血栓形成工具。手性液相色谱-质谱(LC-MS / MS)方法用于评估三种市售LOX抑制剂[七叶皂苷(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(-3,4-肉桂基-α-氰基肉桂酸酯)和乙酰水杨酸(ASA;环氧合酶-1抑制剂)对类前列腺素的生成和使人类全血中的HETEs(羟基二十碳四烯酸)在37°C(血清)下凝结1小时,用胶原蛋白或TRAP-6(一种激活凝血酶的肽激活剂)刺激富血小板血浆(PRP)并洗涤血小板。在血清中,ML-355不会影响类花生酸的产生,而CDC会导致12S-HETE水平的降低不完全。七叶皂甙同时抑制12S-HETE和血栓烷(TX)B 2生产;ASA有选择地影响了TXB 2的生产。在凝血酶,七叶皂苷和CDC刺激的洗涤后的血小板中,抑制12S-HETE和TXB 2均起作用,而ML-355几乎无效。在PRP中,ML-355,CDC和七叶皂甙不影响与类花生酸生物合成作用不完全有关的血小板凝集。ASA单独或与替卡格雷(P2Y 12阻滞剂)联合使用会影响血小板凝集,并严重抑制TXB 2的产生。P2Y 12受体信号传导有助于血小板对12S-HETE生物合成的反应。总之,ML-355,七叶皂苷和CDC在不同细胞系统中不是p-12-LOX的选择性抑制剂。它们不影响胶原蛋白或TRAP-6在PRP中诱导的血小板聚集。对人全血中产生的类花生酸中的12-LOX抑制剂进行表征有助于获得有关其酶选择性,脱靶作用以及血浆成分对其药理作用的可能影响的信息。

更新日期:2020-09-02
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