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Cholesterol enrichment of rabbit platelets enhances the Ca2+ entry pathway induced by platelet-derived secondary feedback agonists

机译:兔血小板中胆固醇的富集增强了血小板衍生的次级反馈激动剂诱导的Ca2 +进入途径

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Aims: Hypersensitivity of platelets due to increased platelet cholesterol levels has been reported in hypercholesterolemia. However, the signaling pathways linking increased platelet reactivity and cholesterol contents are not fully understood. This study aims to determine the direct effect of cholesterol enrichment of platelets on the pathways including Ca2 + mobilization and secondary feedback agonists such as adenosine diphosphate (ADP) and thromboxane A2 (TXA2). Main methods In vitro cholesterol enrichment of rabbit platelets was performed by incubation with cholesterol complexed with methyl-β-cyclodextrin. Ca2 + mobilization was monitored using platelets loaded with fura-PE3/AM, a fluorescent calcium indicator. Released ATP and TXB2 from platelets were measured by a luciferin-luciferase ATP assay system and a TXB2 ELISA Kit, respectively. Key findings Cholesterol enrichment of rabbit platelets significantly enhanced Ca2 + mobilization induced by thrombin, accompanying an augmented Ca2 + entry. The augmentation of Ca 2 + entry by cholesterol enrichment was significantly suppressed by treatment with inhibitors for secondary feedback agonists. In cholesterol-enriched platelets, the amount of released ATP or TXB2 induced by thrombin was not significantly altered in comparison with control platelets, whereas an increase in [Ca2 +]i induced by ADP or U46619, a TXA2 mimetic, was significantly enhanced. Significance These results suggest that cholesterol enrichment of rabbit platelets results in enhanced Ca2 + mobilization via ADP/TXA2-dependent augmentation of the Ca2 + entry pathway. The results reveal a novel mechanism by which platelet hypersensitivity is regulated by cholesterol contents.
机译:目的:在高胆固醇血症中,由于血小板胆固醇水平升高引起的血小板超敏反应已有报道。但是,有关增加血小板反应性和胆固醇含量的信号传导途径尚不完全清楚。这项研究旨在确定血小板中胆固醇富集对包括Ca2 +动员和次级反馈激动剂(如二磷酸腺苷(ADP)和血栓烷A2(TXA2))的直接作用。主要方法通过将胆固醇与甲基-β-环糊精复合,进行体外胆固醇富集。使用装有呋喃-PE3 / AM(一种荧光钙指示剂)的血小板监测Ca2 +的动员。分别通过荧光素-荧光素酶ATP测定系统和TXB2 ELISA试剂盒测量从血小板释放的ATP和TXB2。主要发现兔血小板中胆固醇的富集显着增强了凝血酶诱导的Ca2 +动员,同时增加了Ca2 +的进入。通过使用次级反馈激动剂的抑制剂治疗可显着抑制胆固醇富集引起的Ca 2 +进入的增加。在富含胆固醇的血小板中,与对照血小板相比,凝血酶诱导的释放的ATP或TXB2的量没有明显改变,而由ADP或TXA2模拟物U46619诱导的[Ca2 +] i的增加却显着增强。意义这些结果表明,通过ADP / TXA2依赖性Ca2 +进入途径的增强,兔血小板中胆固醇的富集导致Ca2 +动员的增强。结果揭示了通过胆固醇含量调节血小板超敏性的新机制。

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