ADP overcomes the inhibitory effect of low doses of wortmannin on PtdIns(3,4,5)P3 production, PtdOH synthesis, and platelet aggregation.
Human platelet suspensions were preincubated with or without 10-nmol/L wortmannin (2 minutes, 37°C) and stimulated by FcγRIIA cross-linking in the absence (left panel) or in the presence (right panel) of 5-μmol/L (i) or 10-μmol/L (ii) ADP (A). Aggregation was assessed using a Chrono-Log dual-channel aggregometer with stirring at 900 rev/min. (B,C) 32P-labeled platelets were incubated with 10 or 50-nmol/L wortmannin (2 minutes, 37°C) and activated by FcγRIIA cross-linking in the presence or in the absence of 5-μmol/L or 10-μmol/L ADP as indicated. After 2 minutes of stimulation, lipids were extracted and the radioactivity incorporated in PtdIns(3,4,5)P3 (B) or PtdOH (C) was determined as described in “Materials and methods.” Data are expressed as a percentage of PtdIns(3,4,5)P3 or PtdOH produced compared with control (100% being the production obtained by FcγRIIA clustering at 2 minutes) and are means ± standard errors of 3 independent experiments.
