Abstract
The aggregation of human platelets is an important physiological hemostatic event contingent upon receptor-dependent activation of the surface integrin alphaIIbbeta3 and subsequent binding of fibrinogen. Aggregating platelets form phosphatidylinositol 3, 4-bisphosphate (PtdIns(3,4)P2), which has been reported to stimulate in vitro the activity of the proto-oncogenic protein kinase PKB/Akt, as has phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3). It has been assumed that PtdIns(3,4)P2 is synthesized by either 5-phosphatase-catalyzed hydrolysis of PtdIns(3,4,5)P3 produced by phosphoinositide 3-kinase (PI3K) or phosphorylation by PI3K of PtdIns4P. We investigated the route(s) by which PtdIns(3,4)P2 is formed after directly activating alphaIIbbeta3 with anti-ligand-induced binding site Fab fragment and report that aggregation does not lead to the generation of PtdIns(3,4,5)P3, but to transient formation of PtdIns3P and generation of PtdIns(3,4)P2, the latter primarily by PtdIns3P 4-kinase. Both this novel pathway and the activation of PKB/Akt are inhibited by the PI3K inhibitor, wortmannin, and the calpain inhibitor, calpeptin, constituting the first evidence that PtdIns(3,4)P2 can stimulate PKB/Akt in vivo in the absence of PtdIns(3,4,5)P3. Integrin-activated generation of the second messenger PtdIns(3,4)P2 thus depends upon a route distinct from that known to be utilized initially by growth factors. This pathway is of potential general relevance to the function of integrins.
Highlights
The aggregation of human platelets is an important physiological hemostatic event contingent upon receptordependent activation of the surface integrin ␣IIb3 and subsequent binding of fibrinogen
Platelets can be activated by agents that include agonists for the thrombin receptor (THR-R),1 leading to a change in integrin ␣IIb3 conformation to one that binds plasma fibrinogen (FIB) and results in aggregation
Other work has shown that THR-R-dependent accumulation of PtdIns(3,4)P2 can be impaired by calpeptin, an inhibitor of the Ca2ϩ-dependent protease calpain, which is activated under these conditions (7–9)
Summary
The aggregation of human platelets is an important physiological hemostatic event contingent upon receptordependent activation of the surface integrin ␣IIb3 and subsequent binding of fibrinogen. DiC16PtdIns3P (0.2 mM) was included in incubations, added 3 min after FIB, to determine whether there was platelet lysis resulting in access to PtdIns3P 4-kinase.
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