title: Stereo specific platelet inhibition by the natural LXR agonist 22(R)-OH-cholesterol and its fluorescence labelling with preserved bioactivity and chiral handling in macrophages.
abstract: Two synthetic LXR agonists were recently reported to inhibit collagen-induced platelet aggregation and thrombus formation in mice. We therefore studied whether also natural LXR agonists inhibit human platelet activation and whether they can be fluorescence-labelled preserving their bioactivity for LXR-related functional imaging. The natural LXR agonist 22(R)-OH-cholesterol - but not its stereoisomer 22(S)-OH-cholesterol - inhibited collagen induced platelet shape change and aggregation similar to synthetic LXR agonists in a concentration- and time-dependent manner. First exposure to 22(S)-OH-cholesterol prevented the subsequent inhibition of platelets by 22(R)-OH-cholesterol but not vice versa. 22(R)- and 22(S)-OH-cholesterol could be fluorescence-labelled as 22(R)- and 22(S)-OH-cholesteryl-3-dodecanoic-3-BODIPY esters with high yield and purity using the Steglich acylation. Labelled 22(R)- and 22(S)-OH-cholesterol esters retained the stereo specific bioactivity of their parent compounds, were metabolically stable and not cytotoxic at LXR agonistic concentrations. Live staining with labelled 22(R)- or 22(S)-OH-cholesterol esters demonstrated stereo specific inhibition of platelet spreading and chiral handling by macrophages that reflect LXR activation. The rapid inhibition of platelet reactivity to collagen by natural and pharmacologic LXR agonists offers a mechanism that could attenuate platelet activation by denuded plaques that expose collagen and LXR agonistic oxysterols. Stable fluorescence labelled 22(R)- and 22(S)-OH-cholesterol analogues with preserved stereo specific bioactivity and staining characteristics provide valuable tools for LXR-related functional imaging in pathophysiologic studies, for binding assays and for LXR-targeted drug development.
authors.affiliation: Institute for Cardiovascular Prevention, University of Munich, Pettenkoferstr. 9, 80336 Munich, Germany. Stephanie.Schaffer@med.uni-muenchen.de
authors.forename: Stephanie
authors.initials: S
authors.lastname: Schaffer
authors.forename: Raman
authors.initials: R
authors.lastname: Tandon
authors.forename: Hendrik
authors.initials: H
authors.lastname: Zipse
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Siess
authors.forename: Andreas
authors.initials: A
authors.lastname: Schmidt
authors.forename: Janina
authors.initials: J
authors.lastname: Jamasbi
authors.forename: Ela
authors.initials: E
authors.lastname: Karshovska
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Steglich
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lorenz
mesh_terms: D000215:Acylation; D005456:Fluorescent Dyes; D006801:Humans; D006888:Hydroxycholesterols / Q000494:pharmacology*; D007700:Kinetics; D000071518:Liver X Receptors; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D057093:Orphan Nuclear Receptors / Q000819:agonists*; D015539:Platelet Activation / Q000187:drug effects*; D013237:Stereoisomerism
chemical_list: D005456:Fluorescent Dyes; D006888:Hydroxycholesterols; D000071518:Liver X Receptors; D057093:Orphan Nuclear Receptors; C003585:22-hydroxycholesterol