Collagen related peptide

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.

Formic acid, tert-butyl methyl ether (MTBE), ammonium formate, ammonium acetate, acetate acid (HAc), sodium chloride, sodium bicarbonate, potassium chloride, glucose, disodium phosphate, HEPES, and calcium chloride were purchased from Sigma Aldrich (Steinheim, Germany). The ULC/MS-grade solvents, acetonitrile (ACN), methanol (MeOH) were obtained from Biosolve (Valkenswaard, Netherlands) and isopropanol (IPA) was purchased from Merck (Darmstadt, Germany). Ultrapure water (18 MΩ cm at 25 °C) was used to prepare solutions. Sodium dodecyl sulfate (SDS) was obtained from Roth (Karlsruhe, Germany), tris(hydroxymethyl)-aminomethane (Tris) from Applichem (Darmstadt, Germany), and sodium chloride (NaCl) from Merck (Darmstadt, Germany). Platelets were activated using collagen-related peptide (Richard Farndale, University of Cambridge, United Kingdom) or thrombin from human plasma (Roche, Germany).
One hundred and thirty-six lipid standards (60 mediators, 58 thereof selected after model review) (See Supplementary Methods) were used to study the lipid fragmentation, build optimal collision-energy models, create in silico spectral libraries and spike-in as internal standards. They were purchased from Avanti (Alabaster, AL, USA) and Cayman Chemical (Ann Arbor, Michigan, USA). Lipid fragment masses of mediators were validated with the Metlin database (https://metlin.scripps.edu/)^{66 (link)}.