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. 2004 Feb;113(3):441-50.
doi: 10.1172/JCI20267.

Defects in secretion, aggregation, and thrombus formation in platelets from mice lacking Akt2

Donna Woulfe  1 , Hong Jiang, Alicia Morgans, Robert Monks, Morris Birnbaum, Lawrence F Brass
Affiliations

Defects in secretion, aggregation, and thrombus formation in platelets from mice lacking Akt2

Donna Woulfe et al. J Clin Invest. 2004 Feb.

Abstract

Prior studies have shown that PI3Ks play a necessary but incompletely defined role in platelet activation. One potential effector for PI3K is the serine/threonine kinase, Akt, whose contribution to platelet activation was explored here. Two isoforms of Akt were detected in mouse platelets, with expression of Akt2 being greater than Akt1. Deletion of the gene encoding Akt2 impaired platelet aggregation, fibrinogen binding, and granule secretion, especially in response to low concentrations of agonists that activate the G(q)-coupled receptors for thrombin and thromboxane A(2). Loss of Akt2 also impaired arterial thrombus formation and stability in vivo, despite having little effect on platelet responses to collagen and ADP. In contrast, reducing Akt1 expression had no effect except when Akt2 was also deleted. Activation of Akt by thrombin was abolished by deletion of Galpha(q) but was relatively unaffected by deletion of Galpha(i2), which abolished Akt activation by ADP. From these results we conclude that Akt2 is a necessary component of PI3K-dependent signaling downstream of G(q)-coupled receptors, promoting thrombus growth and stability in part by supporting secretion. The contribution of Akt1 is less evident except in the setting in which Akt2 is absent.

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Figures

Figure 1
Figure 1
Phosphorylation of Akt and GSK-3β in WT, Akt1–/–, and Akt2–/– mice. Washed platelets (2 × 107 platelets in 100 μl) were stimulated for 10 minutes with buffer alone (untxd), thrombin (thr; 1 U/ml), or thrombin in the presence of the PI3K inhibitor, LY294002 (thr + LY; 50 μM) and then immunoblotted with Ab’s directed against (a) Akt phosphorylated on serine 473 (Akt-PSer473), (b) total Akt, (c) GSK-3β phosphorylated on serine 9 (GSK-3β–PSer9), and (d) total GSK-3β. The results shown are representative of those obtained a minimum of three times.
Figure 2
Figure 2
Platelet aggregation. (a) A representative aggregation tracing is shown for each agonist and genotype tested. (b) The maximum extent of aggregation observed in response to increasing doses of AYPGQV was compared in WT versus Akt2–/– mice. Each point is the mean ± SEM of two to six experiments (*P < 0.05 and **P < 0.005 using a paired Student’s t test; number of experiments n is shown in parentheses above each data point). conc, concentration.
Figure 3
Figure 3
Secretion in WT versus Akt-deficient platelets. (a) Dense-granule secretion. Release of (3H)5-HT was determined after incubation with the indicated concentrations of AYPGQV. The results shown are the mean ± SEM of three experiments, *P < 0.05 (Student’s t test). (b) Platelet α-granule secretion. Washed human platelets were stimulated with buffer alone (black line), 250 μM AYPGQV (bold blue line), 500 μM AYPGQV (pink line), or 1M AYPGQV (light blue line), stained with FITC-labeled anti–P selectin Ab, and analyzed by flow cytometry. Similar results were obtained in an additional two experiments. FL1-H, fluorescence intensity.
Figure 4
Figure 4
Fibrinogen binding in WT versus Akt-deficient platelets. Washed human platelets were treated with buffer alone (thin black line), 250 μM AYPGQV (heavy black line), or 500 μM AYPGQV (light gray line) as described in Figure 3b but stained with FITC-labeled mouse fibrinogen (100 μg/ml). The results shown are representative of three experiments.
Figure 5
Figure 5
Thrombotic response of mice to ferric chloride injury of the carotid artery. Flow rates were measured in the carotid artery following exposure to FeCl3. (a) For each genotype, the number of mice forming a stable occlusion (flow rate = 0.1 ml/min) that did not dislodge during the 30-minute time interval are shown in black. The number of mice that formed a transient occlusion that resolved (rate = 0.2 ml/min) is shown in gray, and the number of mice whose arteries remained patent throughout the period of observation is shown in white. (b) Representative flow traces for each genotype.
Figure 6
Figure 6
Tail bleeding times for WT, Akt1–/–, and Akt2–/– mice. Bleeding times were performed as described in Methods. The mean ± SD for each genotype is indicated.
Figure 7
Figure 7
Akt phosphorylation in platelets from Gq–/– and Gi2–/– mice. Washed platelets were obtained and incubated with buffer alone, ADP (10 μM), thrombin (1 U/ml), or thrombin + LY294002 (50 μM) using the protocol described in Figure 1, then immunoblotted with an Ab specific for phosphorylated Akt serine 473. The results shown are representative of those obtained four times each.
Figure 8
Figure 8
Role of Akt in platelet aggregation. Akt is activated in a PI3K-dependent fashion by thromboxane A2 (TxA2) or thrombin. Akt activation promotes secretion of ADP from platelet-dense granules, which stimulates Gi-coupled pathways by binding to the P2Y12 receptor, thereby enhancing aggregate formation and stability.
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References

    1. Trumel C, et al. A key role of adenosine diphosphate in the irreversible platelet aggregation induced by the PAR1-activating peptide through the late activation of phosphoinositide 3-kinase. Blood. 1999;94:4156–4165. - PubMed
    1. Kim S, et al. Protease-activated receptors 1 and 4 do not stimulate G(i) signaling pathways in the absence of secreted ADP and cause human platelet aggregation independently of G(i) signaling. Blood. 2002;99:3629–3636. - PubMed
    1. Dangelmaier C, Jin J, Smith JB, Kunapuli SP. Potentiation of thromboxane A2-induced platelet secretion by Gi signaling through the phosphoinositide-3 kinase pathway. Thromb. Haemost. 2001;85:341–348. - PubMed
    1. Jantzen H-M, Milstone DS, Gousset L, Conley PB, Mortensen RM. Impaired activation of murine platelets lacking G alpha(i2) J. Clin. Invest. 2001;108:477–483. doi:10.1172/JCI200112818. - PMC - PubMed
    1. Li Z, et al. Two waves of platelet secretion induced by thromboxane A2 receptor and a critical role for phosphoinositide 3-kinases. J. Biol. Chem. 2003;278:30725–30731. - PubMed

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