Exogenously added α-synuclein inhibits ionomycin- or thrombin-induced PF4 release from purified platelets.
(A) Ionomycin- and thrombin-induced PF4 release from the α-granules in platelets. Purified platelets (100 μL, more than 106cells/μL) in HEPES-buffered saline containing 0.1% BSA were incubated for 30 minutes on ice to minimize a spontaneous release. Samples were warmed to room temperature for 5 minutes, then 0.5 μM ionomycin or 0.1 U/mL thrombin was added and the reactions were further incubated for 5 minutes at room temperature. Reactions were quenched by placing the samples in ice followed by centrifugation. Supernatants were collected and assayed by quantitative ELISA to determine the amount of PF4. (B) α-Synuclein inhibits ionomycin-induced PF4 release from the α-granules in a dose-dependent manner. (C) α-Synuclein inhibits thrombin-induced PF4 release from the α-granules in a dose-dependent manner. (D) Effect of the incubation time with α-synuclein. Platelet solutions were incubated with 10 μM α-synuclein for the indicated times on ice before stimulation with ionomycin. The PF4 released was quantified using the same method shown in panel A.
