A Phosphoinositide 3-Kinase-AKT-Nitric Oxide-cGMP Signaling Pathway in Stimulating Platelet Secretion and Aggregation

Abstract

Phosphoinositide 3-kinase (PI3K) and Akt play important roles in platelet activation. However, the downstream mechanisms mediating their functions are unclear. We have recently shown that nitric-oxide (NO) synthase 3 and cGMP-dependent protein kinase stimulate platelet secretion and aggregation. Here we show that PI3K-mediated Akt activation plays an important role in agoniststimulated platelet NO synthesis and cGMP elevation. Agonist-induced elevation of NO and cGMP was inhibited by Akt inhibitors and reduced in Akt-1 knock-out platelets. Akt-1 knock-out or Akt inhibitor-treated platelets showed reduced platelet secretion and aggregation in response to low concentrations of agonists, which can be reversed by low concentrations of 8-bromo-cGMP or sodium nitroprusside (an NO donor). Similarly, PI3K inhibitors diminished elevation of cGMP and inhibited platelet secretion and the second wave platelet aggregation, which was also partially reversed by 8-bromo-cGMP. These results indicate that the NO-cGMP pathway is an important downstream mechanism mediating PI3K and Akt signals leading to platelet secretion and aggregation. Conversely, the PI3K-Akt pathway is the major upstream mechanism responsible for activating the NO-cGMP pathway in platelets. Thus, this study delineates a novel platelet activation pathway involving sequential activation of PI3K, Akt, nitric-oxide synthase 3, sGC, and cGMP-dependent protein kinase.Platelets play a critical role in thrombosis and hemostasis. At sites of vascular injury, platelets are activated by various soluble agonists such as thrombin and ADP and adhesive proteins such as collagen and von Willebrand factor. Although different agonists induce platelet activation via different signaling pathways, the signals induced by different agonists converge to common signaling events such as calcium mobilization and activation of the ligand binding function of the integrin ␣ IIb ␤ 3 that mediates platelet aggregation (1, 2). An important feature of platelet activation is the ability to self-amplify the signals, which allows low concentrations of agonists to induce maximal platelet responses. This feature is particularly important in arteries where fast flow of blood may quickly dilute soluble agonists. One important mechanism of selfamplification is the secretion of platelet granule contents such as platelet agonists ADP and serotonin and adhesive proteins von Willebrand factor and fibrinogen (3). The secreted platelet agonists and adhesive proteins, via various pathways, form "positive feedback loops" that greatly amplify and stabilize platelet aggregation, thus sensitizing platelets to low doses of platelet agonists. The signaling mechanism leading to platelet granule secretion is not totally understood. We have recently shown that nitric oxide (NO) 3 synthesized by NO synthase 3 (NOS3, also called eNOS) stimulates soluble guanylyl cyclase and induces cGMP elevation and activation of cGMP-dependent protein kinase (PKG), leading to secretion of platelet granules and the second wave of pla...

Signaling-mediated Functional Activation of Inducible Nitric-oxide Synthase and Its Role in Stimulating Platelet Activation

“…In platelets, it has been known for many years that high concentrations of NO donors (micromolar range) inhibit platelet function (35,36). However, we have shown recently that low concentrations of NO (low nanomolar range) stimulate platelet activation via the cGMPdependent protein kinase pathway, again indicating a biphasic effect of the NO-cGMP pathway in platelet activation (12,13,27,37). It is important to note that the controversy on the role of NO in platelets has been complicated by the fact that investigators in some studies used platelet preparations that are desensitized and thus no longer responsive to low concentrations of agonists (such as 0.1 unit/ml thrombin) (38).…”

“…Agonist-activated platelets generate NO (10,11). We have recently shown that platelet agonists induce activation of phosphoinositide 3-kinase and Akt, which activates eNOS (12) and that eNOS plays an important role in NO synthesis during platelet activation and in stimulating cyclic guanosine monophosphate (cGMP)-dependent platelet secretion and secretion-dependent platelet aggregation (13,14). Here, we show that NO synthesis during platelet activation not only involves eNOS, but is also mediated by the constitutively expressed platelet iNOS.…”

Short‐Term E‐Cigarette Exposure Increases the Risk of Thrombogenesis and Enhances Platelet Function in Mice

“…It was previously shown that phosphorylation of Akt and/or ERK plays a critical role in platelet function,60, 61, 62 and supports thrombus formation 63, 64. Thus, we examined whether e‐cigarettes exert any effect on these 2 markers of platelet activation.…”

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].…”