PHOSPHOINOSITIDE 3-KINASE INHIBITION SPARES ACTIN ASSEMBLY IN ACTIVATING PLATELETS BUT REVERSES PLATELET-AGGREGATION

Platelet stimulation by thrombin leads to the activation of phosphoino sitide 3-kinase (PI 3-K) and to the production of the D3 phosphoinosit ides, phosphatidylinositol 3,4-bisphosphate (PdtIns-3,4-P-2) and 3,4,5 -trisphosphate (PdtIns-3,4,5-P-3). Because changes in the levels of th ese phosphoinositides correlate with the kinetics of actin assembly, t hey have been proposed to mediate actin assembly, causing cell shape c hanges, Wortmannin and LY294002, two unrelated inhibitors of PI 3-K, w ere used to investigate the role of PI 3-K in platelet actin assembly and aggregation. Both PI 3-K inhibitors abrogated the production of Pd tIns-3,4-P-2 and PdtIns-3,4,5-P-3 in thrombin receptor-activating pept ide (TRAP)-stimulated cells. However, neither wortmannin nor LY294002 altered the kinetics of actin assembly or the exposure of nucleation s ites in TRAP-stimulated cells. In contrast, PI 3-K inhibitors showed a specific inhibitory pattern of cell aggregation, characterized by a p rimary phase of aggregation followed by progressive disaggregation. Fl ow cytometry analysis with the PAC1 monoclonal antibody or with FITC-l abeled fibrinogen indicated that wortmannin inhibited the maintenance of the platelet integrin GPIIb-IIIa in its active state. Wortmannin al so inhibited, in a dose-dependent manner, platelet aggregation induced by the binding of the monoclonal antibodies P256 and LIBS-6 to GPIIb- IIIa. LIBS Fab-induced aggregation also led to the production of PdtIn s 3,4 P-2. Platelet secretion, as evidenced by the release of preloade d C-14-5-hydroxytryptamine secretion or P-selectin up-regulation, was not affected by PI 3-K inhibition. These results demonstrate that the generation of D3 phosphoinositides is not required for actin assembly in TRAP-activated platelets, However, PI 3-K stimulation is necessary for prolonged GPIIb-IIIa activation and irreversible platelet aggregat ion, PI 3-K stimulation downstream of GPIIb-IIIa engagement may provid e positive feedback required to sustain active GPIIb-IIIa.