Prostacyclin

Human blood was obtained from consenting donors in accordance with the Declaration of Helsinki under an approved University of Rochester IRB protocol via venipuncture into vacutainer tubes containing 0.105 M sodium citrate (BD, Franklin Lakes, NJ) from healthy donors who had not taken aspirin or other non-steroidal anti-inflammatory drugs for 2 weeks prior to donation. Platelet rich plasma (PRP) was prepared by centrifugation (250 × g for 10 minutes at 20°C). PRP was centrifuged at 1000 × g for 10 minutes at 20°C with 1 μg/mL prostacyclin (Cayman Chemical, Ann Arbor, MI). Platelets were gently resuspended in Tyrode’s (Sigma-Aldrich, St Louis, MO) ACD solution (25/3, vol/vol) containing 0.1 μg/mL prostacyclin, then centrifuged at 1000 × g for 10 minutes. Platelets were resuspended in Tyrode’s and used within 3 hours of collection. Platelets were adjusted to 3 × 10¹⁰ platelets/L for spreading assays or 1 × 10¹¹ platelets/L for all other assays with Tyrode’s. Washed platelets were treated with vehicle (0.1% ethanol) or SPMs (Cayman chemical, >95% pure) for 15 minutes at 20°C then either left unactivated or activated with 5 μM ADP (Chrono-Log Corp., Havertown, PA), 5 μg/mL collagen (Chrono-Log Corp., Havertown, PA), or 0.1 U/mL thrombin for 15–30 minutes at 20°C. Supernatants were generated by centrifugation at 1200 × g for 15 minutes and analyzed for mediator release.

Blood was collected via retro-orbital plexus in 10% final volume of acid-citrate-dextrose anti-coagulant solution (85 mM trisodium citrate dihydrate, 66.6 mM citric acid monohydrate, 111 mM dextrose, 450 mOsm/L, pH 4.5) and diluted with modified Tyrode’s buffer (137 mM NaCl, 11.9 mM NaHCO₃, 0.4 mM Na₂HPO₄, 2.7 mM KCl, 1.1 mM MgCl₂, 5.6 mM glucose, pH 7.3) and centrifuged at 100g for 30 minutes. Platelet-rich plasma and buffy coat were transferred to a new tube and centrifuged at 100g for 20 minutes. Prostacyclin (Sigma Aldrich, St. Louis, MO) was added at 1 μg/mL and apyrase (Sigma Aldrich, St. Louis, MO) was added at 2 U/mL. Platelets were then pelleted at 900g for 7 minutes and resuspended in modified Tyrode’s buffer for quantitation on the Hemavet HV950FS hematology counter and diluted to the appropriate concentration. Platelet aggregation was monitored in a PAP-4 aggregometer equipped with a micro-volume adaptor (Bio/Data Corporation, Horsham, PA) at 37°C for 6 minutes, at 1200 rpm stirring speed, as previously described^{17 (link)}. Aggregation was performed with 180 μl of washed platelets at 1.0 × 10⁸ platelets/mL, added with 20 μL of murine thrombin (Enzyme Research Laboratories, South Bend, IN), acid-soluble calf skin type I collagen (Bio/Data Corp, Horsham), and adenosine diphosphate (ADP) (Bio/Data) with doses indicated in Figures.