Search

The Akt family of serine/threonine kinases includes Akt1, Akt2, and Akt3 isoforms. Prior studies have reported that Akt1 and Akt2, but not Akt3, are expressed in platelets. Here, we show that Akt3 is expressed in substantial amounts in platelets. Akt3−/− mouse platelets selectively exhibit impaired platelet aggregation and secretion in response to low concentrations of thrombin receptor agonists and thromboxane A2 (TXA2), but not collagen or VWF. In contrast, platelets from Akt1−/− or Akt2−/− mice are defective in platelet activation induced by thrombin, TXA2, and VWF, but only Akt1−/− platelets show significant defects in response to collagen, indicating differences among Akt isoforms. Akt3−/− platelets exhibit a significant reduction in thrombin-induced phosphorylation of glycogen synthase kinase 3β (GSK-3β) at Ser9, which is known to inhibit GSK-3β function. Thus, Akt3 is important in inhibiting GSK-3β. Accordingly, treatment of Akt3−/− platelets with a GSK-3β inhibitor rescued the defect of Akt3−/− platelets in thrombin-induced aggregation, suggesting that negatively regulating GSK-3β may be a mechanism by which Akt3 promotes platelet activation. Importantly, Akt3−/− mice showed retardation in FeCl3-induced carotid artery thrombosis in vivo. Thus, Akt3 plays an important and distinct role in platelet activation and in thrombosis.

Current antithrombotic drugs have an adverse effect on bleeding, highlighting the need for new molecular targets for developing antithrombotic drugs that minimally affect hemostasis. Here we show that LIMK1(-/-) mice have defective arterial thrombosis in vivo but do not differ from wild-type mice with respect to bleeding time. LIMK1(-/-) mice show a selective defect in platelet activation induced through the von Willebrand Factor (VWF) receptor, the glycoprotein Ib-IX-V complex (GPIb-IX), but not by GPIb-IX-independent platelet agonists. In fact, LIMK1(-/-) platelets show an enhanced reaction to certain GPIb-IX-independent agonists. The defect of LIMK1(-/-) platelets in GPIb-IX-mediated platelet activation is attributed to a selective inhibition in VWF/GPIb-IX-induced phosphorylation of cytosolic phospholipase A2 (cPLA2) and consequent thromboxane A2 (TXA2) production. Supplementing a TXA2 analog, U46619, corrected the defect of LIMK1(-/-) platelets in VWF-induced stable platelet adhesion. Although LIMK1(-/-) platelets also showed reduced actin polymerization after GPIb-IX-mediated platelet aggregation, actin polymerization inhibitors did not reduce TXA2 generation, but rather accelerated platelet aggregation, suggesting that the role of LIMK1 in GPIb-mediated platelet activation is independent of actin polymerization. Thus, LIMK1 plays a novel role in selectively mediating GPIb-IX-dependent TXA2 synthesis and thrombosis and represents a potential target for developing antithrombotic drugs with minimal bleeding side effect.

Antagonists of platelet integrin alphaIIbbeta3 are potent anti-thrombotics due to critical roles of integrins in thrombosis. However, integrins are also important in hemostasis, and thus integrin antagonists have potentially life-threatening bleeding side effect. It would be ideal if we can develop integrin antagonists without bleeding side effect. Integrins transmit signals bidirectionally. Intracellular signals activate integrin alphaIIbbeta3, leading to talin-dependent integrin ligation, which is required for platelet adhesion and initial hemostatic thrombus formation. Integrin ligation in turn mediates Galpha13/Src-dependent outside-in signaling that stabilizes and amplify thrombi, which is crucial for occlusive arterial thrombosis. Here we show that talin and Galpha13 bind to mutually exclusive but distinct sites in integrin beta3, and their bindings are dynamically regulated during integrin signaling. The first talin binding wave mediates inside-out signaling and also “ligand-induced integrin activation”, but is not required for early phase outside-in signaling. Integrin ligation induces talin dissociation and Galpha13 binding, which selectively mediates early phase outside-in signaling. The second talin binding wave is associated with late phase outside-in signaling and clot retraction. Based on these findings, we have designed a selective inhibitor of Galpha13-integrin interaction, which specifically abolished outside-in signaling without affecting inside-out signaling and integrin ligation. Strikingly, this inhibitor potently inhibits occlusive thrombosis in vivo, but has no effect on tail bleeding time, in contrast to the current integrin antagonists. Thus, we have discovered a mechanism for switching the direction of integrin signaling and a new anti-thrombotic that does not cause bleeding. Disclosures: No elevant conflicts of interest to declare.

Abstract 1134 Ligand binding to integrins mediates cell adhesion and transmits “outside in” signals that lead to cell spreading, migration, and proliferation. In platelets, the prototype integrin aIIbb3-mediated outside-in signaling is required for platelet spreading and retraction, and greatly amplifies platelet activation. Previous studies suggest that phosphoinositide 3-Kinases (PI3K) are activated upon binding of integrin αIIbβ3 to its ligand fibrinogen, and is important in outside-in signaling leading to platelet spreading. However, the mechanism by which PI3K transmits outside-in signals has been unclear. A major known downstream effector of PI3K is the Akt (protein kinase B) family of serine/threonine kinases, including Akt1, Akt2, and Akt3. We have recently shown that platelets not only express Akt1 and Akt2 as previously reported, but also express a substantial amount of Akt3. To investigate whether Akt3 is a downstream effector mediating PI3K-dependent integrin outside-in signaling, platelets from Akt3 knockout mice were compared with wild type platelets for their spreading on fibrinogen. Platelets from Akt3−/− mice showed partially, but significantly reduced spreading on fibrinogen, indicating that Akt3 is important in integrin-mediated outside-in signaling leading to platelet spreading. Consistent with the results of Akt3 knockout, treatment of platelets with a pan Akt inhibitor also significantly inhibited spreading of human and mouse platelets on fibrinogen. Akt becomes phosphorylated upon platelet spreading on fibrinogen, which is significantly reduced in Akt3 knockout platelets, and is abolished by PI3 Kinase inhibitor, wortmannin, or Src Family Kinase (SFK) Inhibitor, PP2, suggesting that Akt activation is downstream from PI3K, and SFK during integrin outside-in signaling. Thus, our data reveals that Akt3 is an important downstream effector of PI3K-dependent integrin outside-in signaling promoting platelet spreading. Disclosures: No relevant conflicts of interest to declare.

Akt is a family of serine/threonine kinases that are activated downstream of Phosphoinositide 3 Kinases (PI3K). There are three known Akt isoforms: Akt1, Akt2, and Akt3. Both Akt1 and Akt2 have been shown to be expressed in platelets and play important roles in PI3K-dependent platelet activation signaling. Previous studies have concluded that Akt3 is not expressed in platelets; however, we show that Akt3 is in fact a major Akt isoform present in platelets, and demonstrate that Akt3 plays an important role in platelet activation. In order to determine if Akt3 mRNA is present in platelets, RNA was isolated from platelets and RT-PCR experiments were performed using Akt3 specific primers. Akt3 mRNA was detected in both human and mouse platelets, and the possibility that Akt3 mRNA came from leukocyte contamination was excluded because Akt3 was not detected with RT-PCR using RNA purified from the same number of leukocytes as that in platelet preparation. To assess if Akt3 protein is present in platelets, western blot analysis was performed using an Akt3 specific antibody. Results revealed that Akt3 is highly expressed in human and mouse platelets. In order to assess the relative amount of Akt3 present in platelets, total Akt levels and phosphorylated Akt levels were measured in Akt3 knockout mouse platelets in comparison with wild type mouse platelets. Akt3 knockout mouse platelets showed a significant decrease in total Akt, and also a significant reduction in phosphorylated Akt, as indicated by Western blot analysis of phospho-Thr308-Akt and phospho-Ser473-Akt antibodies. Thus, our data indicates Akt3 is a major Akt isoform expressed in platelets. To determine the role of Akt3 in platelet activation, platelets from wild type mice and Akt3 knockout mice were stimulated with low concentrations of platelet agonists. Akt3 knockout mouse platelets exhibited impaired aggregation and ATP secretion in response to low dose thrombin and collagen compared with wild type controls, indicating that Akt3 plays an important role in promoting platelet activation. In order to assess the signaling molecules downstream from Akt3, Akt3 knockout and wild type mouse platelets were stimulated with low dose thrombin and phosphorylation of GSK-3β, an enzyme recently reported to negatively regulate platelet function downstream from Akt1 or Akt2 in platelets, was assessed. Western blot for phospho-Ser9 of GSK-3β in Akt3 knockout mouse platelets stimulated with thrombin showed less phosphorylation compared to Akt1 or Akt2 knockout mouse platelets, indicating that Akt3 is the major Akt isoform responsible for GSK-3β phosphorylation. Thus, our data shows that Akt3 is a major Akt isoform in platelets and plays an important role in platelet activation. Since phosphorylation of GSK-3β at Ser9 has been reported to reverse the inhibitory effect of GSK-3β on platelet function, our data also suggest that the stimulatory role of Akt3 in platelet activation may be mediated by negative regulation of GSK-3β function.