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

Abstract

AimsHypersensitivity 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 methodsIn 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 findingsCholesterol enrichment of rabbit platelets significantly enhanced Ca2+ mobilization induced by thrombin, accompanying an augmented Ca2+ entry. The augmentation of Ca2+ 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. SignificanceThese 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.