The role of Akt in the signaling pathway of the glycoprotein Ib-IX–induced platelet activation

Abstract

The platelet von Willebrand factor (vWF) receptor, glycoprotein Ib-IX (GPIb-IX), mediates platelet adhesion and induces signaling leading to integrin activation. Phosphoinositol 3-kinase (PI3K) is important in GPIb-IX–mediated signaling. PI3K–dependent signaling mechanisms, however, are unclear. We show that GPIb-IX–induced platelet aggregation and stable adhesion under flow were impaired in mouse platelets deficient in PI3K effectors, Akt1 and Akt2, and in human platelets treated with an Akt inhibitor, SH-6. Akt1 and Akt2 play important roles in early GPIb-IX signaling independent of Syk, adenosine diphosphate (ADP), or thromboxane A2 (TXA2), in addition to their recognized roles in ADP- and TXA2–dependent secondary amplification pathways. Knockout of Akt1 or Akt2 diminished platelet spreading on vWF but not on immobilized fibrinogen. Thus, Akt1 and Akt2 are both required only in the GPIb-IX–mediated integrin activation (inside-out signaling). In contrast, PI3K inhibitors abolished platelet spreading on both vWF and fibrinogen, indicating a role for PI3K in integrin outside-in signaling distinct from that in GPIb-IX–mediated inside-out signaling. Furthermore, Akt1- or Akt2-deficiency diminished vWF–induced cGMP elevation, and their inhibitory effects on GPIb-IX–dependent platelet adhesion were reversed by exogenous cGMP. Thus, Akt1 and Akt2 mediate GPIb-IX signaling via the cGMP–dependent signaling pathway.

ARQ 092, an orally-available, selective AKT inhibitor, attenuates neutrophil-platelet interactions in sickle cell disease

“…Although oral administration of hydroxyurea or ARQ 092 alone did not affect intracellular ROS generation in fMLP-stimulated neutrophils isolated from the mice, co-administration of hydroxyurea and ARQ 092 significantly decreased ROS generation, suggesting that combined therapy may efficiently attenuate oxidative stress conditions in SCD patients. Consistent with previous reports showing that the activation and adhesive function of platelets is regulated by AKT, [17][18][19]36 we found that ARQ 092 significantly impairs P-selectin exposure and GPIbα-mediated agglutination in platelets from SCD patients in vitro and in SCD mouse platelets ex vivo. In support of the importance of neutrophil αMb2 integrin and platelet P-selectin and GPIbα for neutrophil-platelet interactions, 13 we observed that specific AKT inhibition in both neutrophils and platelets isolated from SCD patients effectively decreases heterotypic cell-cell aggregation.…”

PKC inhibitors RO 31-8220 and Gö 6983 enhance epinephrine-induced platelet aggregation in catecholamine hypo-responsive platelets by enhancing Akt phosphorylation

“…This should be further investigated in relation to future advancements in specific agonists and antagonists of each PKC isoform. Epinephrine-mediated α2A-adrenoceptor-coupled Gz signaling alone is known to be incapable of inducing full platelet aggregation, and activation of P2Y12 receptor-coupled Gi signaling, which leads to inhibition of adenylyl cyclase, is required (22)(23)(24). An elevated plasma level of NO has been observed in CA-HY compared to CA-GR (10), and NO is known to raise cAMP levels in platelets (25).…”

An Important Role of the Src Family Kinase Lyn in Stimulating Platelet Granule Secretion

“…The role of SFK (Lyn) as well as the cGMP pathway in GPIb-IX signaling appears to be proximal to the receptor and is important in the stable platelet adhesion to von Willebrand factor under shear stress, independent of ADP secretion (17,32,33). It is recognized that SFK is important in the collagen-induced activation of the ITAM pathway because SFK mediates tyrosine phosphorylation of the ITAM motif of FcR␥ required for recruiting the downstream kinase Syk, which is required for collagen-induced platelet secretion and aggregation.…”

“…Furthermore, PI3K and Akt are known to promote platelet secretion and secretion-dependent platelet aggregation (16,20,53,54), and the effects of PI3K and Akt are mediated via the NO-sGC/cGMP-PKG pathway (20,32). In this study, we further show that 1) the SFK inhibitor, PP2, inhibited agonist-induced Akt phosphorylation, indicating that SFK is important in GPCR-mediated activation of the PI3K-Akt signaling pathway; 2) PP2 and Lyn knock-out also inhibited thrombin-and collagen-induced increases in the intracellular cGMP levels in platelets, indicating that SFK is upstream of the NO-cGMP signaling pathway; 3) supplementation of low concentrations of 8-bromo-cGMP corrected the platelet aggregation defect caused by PP2 and Lyn knock-out and partially but significantly reversed the inhibitory effect of PP2 on platelet granule secretion, indicating that SFK (Lyn)-mediated activation of the cGMP signaling pathway plays an important role in the stimulatory effect of SFK (Lyn) on platelet secretion and aggregation; and 4) similar to PP2, the specific sGC inhibitor ODQ inhibited platelet aggregation in response to low dose thrombin (19).…”