Impaired platelet responses to thrombin and collagen in AKT-1–deficient mice

Abstract

We investigated the role of Akt-1, one of the major downstream effectors of phosphoinositide 3-kinase (PI3K), in platelet function using mice in which the gene for Akt-1 had been inactivated. Using ex vivo techniques, we showed that Akt-1-deficient mice exhibited impaired platelet aggregation and spreading in response to various agonists. These differences were most apparent in platelets activated with low concentrations of thrombin. Although Akt-1 is not the predominant Akt isoform in mouse platelets, its absence diminished the amount of total phospho-Akt and inhibited increases in intracellular Ca 2؉ concentration in response to thrombin. Moreover, thrombin-induced platelet ␣-granule release as well as release of adenosine triphosphate from dense granules was also defective in Akt-1-null platelets. Although the absence of Akt-1 did not influence expression of the major platelet receptors for thrombin and collagen, fibrinogen binding in response to these agonists was significantly reduced. As a consequence of impaired ␣ IIb ␤ 3 activation and platelet aggregation, Akt-1 null mice showed significantly longer bleeding times than wild-type mice. IntroductionUnder normal conditions, platelets circulate freely in the blood without interacting with each other or the vessel wall. On vascular injury, subendothelial matrix proteins, including collagens, or soluble agonists trigger platelet activation. The hallmark of platelet activation is the transformation of the major platelet glycoprotein, ␣ IIb ␤ 3 , from its resting to active state, which serves as a fibrinogen receptor, thereby mediating platelet aggregation. One of the most potent platelet agonists, thrombin, acts via a dual system of G protein-coupled protease-activated receptors, PAR3 and PAR4. Both of these receptors are required for optimal thrombin-induced aggregation and secretion. 1 The majority of platelet agonists, including thrombin and collagen, activate phosphoinositide 3-kinase (PI3K) in platelets. Inhibitors of PI3K (wortmannin and LY294002) block fibrinogen binding and platelet aggregation induced by thrombin and collagen, indicating a role for PI3K in ␣ IIb ␤ 3 activation. 2 Platelets contain 2 major forms of PI3K, p85/p110 PI3K, composed of a p110 catalytic and p85 regulatory subunit and PI3K␥, composed of a p110␥ catalytic and p101 regulatory subunit. Both forms of PI3K are involved in the inside-out signaling that activates ␣ IIb ␤ 3 and induces platelet aggregation. 2 Recent studies demonstrated that a deficiency in the p85␣ regulatory subunit in mice leads to a significant reduction of collagen-induced platelet aggregation, particularly at low doses of stimulus. . 3 The absence of PI3K␥ activity, indicated by the lack of Akt phosphorylation, leads to impaired platelet aggregation in response to adenosine diphosphate (ADP) and protects against thrombosis. 4 PI3Ks generate phosphoinositide products that target the Tec family tyrosine kinases, serine/threonine protein kinases such as Akt, guanosine diphosphate/guanosine triphosphate exchange f...

Altered nitric oxide/cGMP platelet signaling pathway in platelets from patients with acute coronary syndromes

“…PI3K has been observed to promote Akt activation during platelet stimulation [16,25], and two different isoforms of Akt, Akt1/PKBα and Akt2/PKBβ, have been found to play a role in platelet activation [3]. Once phosphorylated, Akt is known to phosphorylate and activate in its turn other proteins, including eNOS [9].…”

A Platelet Secretion Pathway Mediated by cGMP-dependent Protein Kinase

“…In this respect, it is interesting to note that endothelial nitric oxide synthase can be activated in endothelial cells by Akt (50), a serine/threonine protein kinase that is a downstream effector of phosphatidylinositol 3-kinase. We and others have shown that phosphatidylinositol 3-kinase and Akt play important roles in platelet secretion and second wave platelet aggregation (12,51,52). Thus, it will be interesting to investigate further whether activation of cGMP/PKG is downstream from the phosphatidylinositol 3-kinase-Akt pathway during platelet activation.…”

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

“…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).…”