Fig. 2.

Stimulation of membrane 5-hydroxytryptamine receptor (5-HT2AR) induces tumor necrosis factor-alpha-converting enzyme (TACE)-mediated glycoprotein (GP)Ibα shedding. (A) Isolated wild-type (WT) platelets were incubated with 100 µm 5-HT, 20 µm fluoxetine (Flx), 20 µm ketanserin, 50 µm (−)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) or dimethylsulfoxide (DMSO) (vehicle) as indicated, and surface GPIbα was measured by flow cytometry (n ≥ 8 mice). (B) Dose-dependency (60-min incubation) and (C) time-dependency (100 µm 5-HT) of GPIbα decrease with 5-HT and 20 µm fluoxetine (n = 5). (D) GPIbα levels after 20 µm fluoxetine/50 µm DOI (n = 8) incubation with or without TACE inhibition by 5 µm tumor necrosis factor-alpha protease inhibitor (TAPI-1). (E) Glycocalicin in supernatant of wild-type platelets after incubation with 20 µm fluoxetine and 5-HT at the indicated concentrations, 300 nm phorbol 12-myristate 13-acetate (PMA), and 5 µm TAPI-1 (representative of three western blot analyses; other blots showed a stronger band after fluoxetine than after vehicle treatment). Below, GPIbα in lysed platelets from the same samples. ***P < 0.001 as compared with wild type/vehicle. MFI, mean fluorescence intensity.