We have shown previously that ADP released upon platelet adhesion mediated by α(IIb)β3 integrin triggers accumulation of phosphatidylinositol 3',4'-bisphosphate (PtdIns-3,4-P2) (Gironcel, D., Racaud-Sultan, C., Payrastre, B., Haricot, M., Borchert, G., Kieffer, N., Breton, M., and Chap, H. (1996) FEBS Lett. 389, 253-256). ADP has also been involved in platelet spreading. Therefore, in order to study a possible role of phosphoinositide 3-kinase in platelet morphological changes following adhesion, human platelets were pretreated with specific phosphoinositide 3- kinase inhibitors LY294002 and wortmannin. Under conditions where PtdIns- 3,4-P2 synthesis was totally inhibited (25 μM LY294002 or 100 nM wortmannin), platelets adhered to the fibrinogen matrix, extended pseudopodia, but did not spread. Moreover, addition of ADP to the medium did not reverse the inhibitory effects of phosphoinositide 3-kinase inhibitors on platelet spreading. Although synthetic dipalmitoyl PtdIns-3,4-P2 and dipalmitoyl phosphatidylinositol 3',4',5'-trisphosphate restored only partially platelet spreading, phosphatidylinositol 4',5'-bisphosphate (PtdIns-4,5-P2) was able to trigger full spreading of wortmannin-treated adherent platelets. Following 32P labeling of intact platelets, the recovery of [32P]PtdIns-4,5-P2 in anti-talin immunoprecipitates from adherent platelets was found to be decreased upon treatment by wortmannin. These results suggest that the lipid products of phosphoinositide 3-kinase are required but not sufficient for ADP-induced spreading of adherent platelets and that PtdIns-4,5-P2 could be a downstream messenger of this signaling pathway.
