Wortmannin and LY294002 partially inhibit collagen-induced formation of inositol phosphates and phosphatidic acid.

(A) [³H]Inositol labeled platelets resuspended at 4 × 10⁸/mL were incubated with different concentrations of either LY294002 (i) or wortmannin (ii) for 3 to 15 minutes before stimulation with 10 μg/mL collagen (s) or 0.1 U/mL thrombin (l) for 5 minutes. Results are expressed as percentage (%) of the response to collagen or thrombin (= 100% in each case) from 1 experiment made in quadruplicate, which is representative of 4 experiments. The mean ± SEM basal dpm was 101 ± 12.4, and maximum stimulations over basal were 1019 ± 133.2 (collagen) and 954 ± 8.4 (thrombin). (B) [³²P]orthophosphate-labeled platelets were stimulated with collagen (10 μg/mL) for 2 minutes and phosphatidic acid extracted as described in “Materials and methods.” LY294002 (LY) or wortmannin (Wt) for 3 to 15 minutes before stimulation with collagen. Results are shown as mean ± SEM percentage of the increase in phosphatidic acid by collagen over basal from 3 experiments.