Tj. Kovacsovics et al., PHOSPHOINOSITIDE 3-KINASE INHIBITION SPARES ACTIN ASSEMBLY IN ACTIVATING PLATELETS BUT REVERSES PLATELET-AGGREGATION, The Journal of biological chemistry, 270(19), 1995, pp. 11358-11366
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.