PLATELET ACTIVATING FACTOR AND SHEEP PLATELETS - A SENSITIVE NEW BIOASSAY

The in vitro response of sheep platelets to platelet activating factor (PAF) was investigated. Sheep platelet-rich plasma aggregated in response to PAF with an EC50 of 10 nM. Platelets isolated via arabinogalactan density gradient centrifugation displayed an EC50 of 50 nM with a threshold response at 0.1 pM. PAF-induced release of 14C-serotonin from isolated sheep platelets was comparable with an EC50 of 50 pM and threshold release at 10 fM. PAF-induced aggregation was specific in that it could be blocked by the competitive receptor antagonists Alprazolam (Upjohn, IC50 = 40 μM), L-652, 731 (MSD, IC50 = 6 μM), and WEB 2086 (Boehringer Ingelheim, IC50 = 0.8 μM). At micromolar concentrations, WEB 2086 did not inhibit ADP- or thrombin-induced aggregation nor thrombin-induced serotonin release. However, at higher concentrations of WEB 2086 some inhibition of thrombin-induced platelet aggregation and release was observed. Subsequent experiments demonstrated that high concentrations of WEB 2086 can inhibit thrombin-induced clotting (Ki = 866 μM) and cleavage of the chromogenic substrate Spectrozyme-TH (Ki = 842 μM). In summary, the response of isolated sheep platelets to PAF was specifically inhibitable and was 10 to 100 times more sensitive than washed rabbit platelets, the most popular bioassay currently in use. © 1990.