Cholesterol enrichment of rabbit platelets enhances the Ca(2+) entry pathway induced by platelet-derived secondary feedback agonists.

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 Ca(2+) mobilization and secondary feedback agonists such as adenosine diphosphate (ADP) and thromboxane A2 (TXA2). In vitro cholesterol enrichment of rabbit platelets was performed by incubation with cholesterol complexed with methyl-β-cyclodextrin. Ca(2+) 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. Cholesterol enrichment of rabbit platelets significantly enhanced Ca(2+) mobilization induced by thrombin, accompanying an augmented Ca(2+) 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 [Ca(2+)]i induced by ADP or U46619, a TXA2 mimetic, was significantly enhanced. These results suggest that cholesterol enrichment of rabbit platelets results in enhanced Ca(2+) mobilization via ADP/TXA2-dependent augmentation of the Ca(2+) entry pathway. The results reveal a novel mechanism by which platelet hypersensitivity is regulated by cholesterol contents.