Isolation and Activation of Murine Platelets

Platelets were prepared as described previously [40 (link),41 (link),42 (link),43 (link),44 (link),45 (link),46 (link)]. Platelets were obtained from 10- to 12-week-old mice of either sex. The mice were anesthetized, and 800 µL blood was drawn from the retro-orbital plexus into tubes with 200 µL acid-citrate-dextrose buffer before the mice were sacrificed [47 (link)]. Platelet-rich plasma (PRP) was obtained by centrifugation at 260 g for 5 min. Afterwards, PRP was centrifuged at 640 g for 5 min to pellet the platelets. Where necessary, apyrase (0.02 U/mL; Sigma-Aldrich) and prostaglandin I₂ (0.5 µM; Calbiochem, Darmstadt, Germany) were added to the PRP to prevent the activation of platelets during isolation [48 ]. After two washing steps, the pellet of washed platelets was resuspended in modified Tyrode-HEPES buffer (pH 7.4, supplemented with 1 mM CaCl₂). Where indicated, collagen-related peptide (Roche, Basel, Switzerland) or thrombin (Roche, Basel, Switzerland) were added at the indicated concentrations [49 (link)]. Platelets have been pre-incubated with 0–33 µM garcinol (R&D, Wiesbaden, Germany) for 30 min at 37 °C before stimulation.