Phosphoinositide 3-kinase inhibition spares actin assembly in activating platelets but reverses platelet aggregation.
Platelet stimulation by thrombin leads to the activation of phosphoinositide 3-kinase (PI 3K) and to the production of the D3 phosphoinositides, phosphatidylinositol 3,4-bisphosphate (PdtIns-3,4P2) and 3,4,5-trisphosphate (PdtIns-3,4,5-P3). Because changes in the levels of these phosphoinositides correlate with the kinetics of actin assembly, they have been proposed to mediate actin assembly, causing cell shape changes. Wortmannin and LY294002, two unrelated inhibitors of PI 3-K, were used to investigate the role of PI 3-K in platelet actin assembly and aggregation. Both PI 3-K inhibitors abrogated the production of PdtIns-3,4-P2 and PdtIns-3,4,5-P3 in thrombin receptor-activating peptide (TRAP)-stimulated cells. However, neither wortmannin nor LY294002 altered the kinetics of actin assembly or the exposure of nucleation sites in TRAP-stimulated cells. In contrast, PI 3-K inhibitors showed a specific inhibitory pattern of cell aggregation, characterized by a primary phase of aggregation followed by progressive disaggregation. Flow cytometry analysis with the PAC1 monoclonal antibody or with FITC-labeled fibrinogen indicated that wortmannin inhibited the maintenance of the platelet integrin GPIIb-IIIa in its active state. Wortmannin also 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 PdtIns-3,4-P2. Platelet secretion, as evidenced by the release of preloaded 14C-5-hydroxy-tryptamine 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 aggregation. PI 3-K stimulation downstream of GPIIb-IIIa engagement may provide positive feedback required to sustain active GPIIb-IIIa.
Kovacsovics
T J
TJ
Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, USA.
Bachelot
C
C
Toker
A
A
Vlahos
C J
CJ
Duckworth
B
B
Cantley
L C
LC
Hartwig
J H
JH
D000199:Actins / Q000096:biosynthesis* / Q000097:blood; D000730:Androstadienes / Q000494:pharmacology; D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D002867:Chromones / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D009025:Morpholines / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D010710:Phosphates / Q000097:blood; D019869:Phosphatidylinositol 3-Kinases; D010743:Phospholipids / Q000096:biosynthesis / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors* / Q000097:blood; D010974:Platelet Aggregation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000187:drug effects / Q000502:physiology; D000077191:Wortmannin
D000199:Actins; D000730:Androstadienes; D000911:Antibodies, Monoclonal; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D010446:Peptide Fragments; D010710:Phosphates; D010743:Phospholipids; D010761:Phosphorus Radioisotopes; D010980:Platelet Membrane Glycoproteins; C082835:thrombin receptor peptide (42-47); C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D000077191:Wortmannin