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

Woulfe, Donna S.; Jiang, Hong; Morgans, Alicia K.; Monks, Robert; Birnbaum, Morris J.; Brass, Lawrence F.

doi:10.1172/jci200420267

Cited by 202 publications

(249 citation statements)

References 39 publications

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“…Although high concentrations of NO donors inhibit platelet activation, endogenous NO production in platelets occurs only when platelets are stimulated with various platelet agonists. Furthermore, potential NOS3 activation signals suggested in other cell types, namely, calcium elevation and the phosphoinositide 3-kinase-Akt pathway (42), all play roles in stimulating platelet activation but not in inhibiting platelet activation (22,(43)(44)(45)(46). Thus, our data that NO in fact plays biphasic roles in platelet activation provide an explanation for this apparent contradiction.…”

Section: Discussionmentioning

confidence: 66%

“…It is important to note that we have recently found that phosphoinositide 3-kinase, like NOS3, plays a critical role in aggregation-dependent platelet secretion and secretiondependent second wave platelet aggregation (22). Others have reported a stimulatory role for Akt in platelet secretion and aggregation (45,46). Also, calcium elevation is induced by platelet agonists and aggregationdependent integrin outside-in signaling (47).…”

Section: Discussionmentioning

confidence: 95%

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Stimulatory Roles of Nitric-oxide Synthase 3 and Guanylyl Cyclase in Platelet Activation

Marjanovic

Stojanović

et al. 2005

Journal of Biological Chemistry

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Nitric oxide (NO) stimulates soluble guanylyl cyclase and, thus, enhances cyclic guanosine monophosphate (cGMP) levels. It is a currently prevailing concept that NO inhibits platelet activation. This concept, however, does not fully explain why platelet agonists stimulate NO production. Here we show that a major platelet NO synthase (NOS) isoform, NOS3, plays a stimulatory role in platelet secretion and aggregation induced by low doses of platelet agonists. Furthermore, we show that NOS3 promotes thrombosis in vivo. The stimulatory role of NOS is mediated by soluble guanylyl cyclase and results from a cGMP-dependent stimulation of platelet granule secretion. These findings delineate a novel signaling pathway in which agonists sequentially activate NOS3, elevate cGMP, and induce platelet secretion and aggregation. Our data also suggest that NO plays a biphasic role in platelet activation, a stimulatory role at low NO concentrations and an inhibitory role at high NO concentrations.Development of thrombotic diseases involves the injury or dysfunction of the blood vessel wall and activation of blood platelets (1). Upon exposure to agonists such as thrombin, ADP, collagen, and von Willebrand factor (VWF), 2 platelets become "activated" and aggregate to form primary thrombi (1, 2). Activated platelets secrete large quantities of ADP, serotonin, and other factors that amplify platelet activation and stabilize platelet aggregates (3). Activated platelets also secrete pro-coagulation, pro-inflammatory, and growth factors (3-5). Thus, platelet activation plays a major role not only in acute arterial thrombosis but also in the development of chronic vascular diseases, such as atherosclerosis, which in turn causes thrombosis (1, 6).A major advance in the field of vascular biology in the last century was the discovery of the vessel dilator, nitric oxide (NO) (7-9). NO is a short-lived messenger molecule synthesized from L-arginine by a family of enzymes known as nitric-oxide synthases (NOS). Three isoforms of NOS enzymes are known (10 -12): NOS1 (neuronal NOS), NOS2 (inducible NOS), and NOS3 (endothelial NOS). NOS3 is the major isoform known to be expressed in platelets (13). One of the major functions of NO is to stimulate soluble guanylyl cyclase (sGC) and increase the synthesis of cyclic guanosine monophosphate (cGMP) that serves as a secondary messenger regulating the function of cGMP-dependent protein kinase (PKG), cGMP-dependent ion channels, and cGMP-regulated phosphodiesterases (7). High concentrations of NO can also chemically modify (nitrosylation and nitration) proteins and, thus, affect cell functions in a cGMP-independent manner (7, 14 -16). NO is involved in diverse processes, such as smooth muscle relaxation, neurotransmission, immune responses, and inflammation (7). It has been a prevailing concept that NO, by elevating intracellular cGMP, inhibits platelet activation (8). This concept is supported by data that high concentrations of NO donor compounds inhibit platelet activation (17-19). However, the concept...

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Section: Discussionmentioning

confidence: 66%

Section: Discussionmentioning

confidence: 95%

Stimulatory Roles of Nitric-oxide Synthase 3 and Guanylyl Cyclase in Platelet Activation

Marjanovic

Stojanović

et al. 2005

Journal of Biological Chemistry

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“…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.…”

Section: Discussionmentioning

confidence: 96%

A Platelet Secretion Pathway Mediated by cGMP-dependent Protein Kinase

Zhang

Marjanovic

et al. 2004

Journal of Biological Chemistry

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Platelet secretion (exocytosis) is critical in amplifying platelet activation, in stabilizing thrombi, and in arteriosclerosis and vascular remodeling. The signaling mechanisms leading to secretion have not been well defined. We have shown previously that cGMP-dependent protein kinase (PKG) plays a stimulatory role in platelet activation via the glycoprotein Ib-IX pathway. Here we show that PKG also plays an important stimulatory role in mediating aggregation-dependent platelet secretion and secretion-dependent second wave platelet aggregation, particularly those induced via G q -coupled agonist receptors, the thromboxane A2 (TXA2) receptor, and protease-activated receptors (PARs). PKG I knock-out mouse platelets and PKG inhibitor-treated human platelets showed diminished aggregation-dependent secretion and also showed a diminished secondary wave of platelet aggregation induced by a TXA2 analog and thrombin receptor-activating peptides that were rescued by the granule content ADP. Low dose collageninduced platelet secretion and aggregation were also reduced by PKG inhibitors. Furthermore PKG I knockout and PKG inhibitors significantly attenuated activation of the G i pathway that is mediated by secreted ADP. These data unveil a novel PKG-dependent platelet secretion pathway and a mechanism by which PKG promotes platelet activation.

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“…These agonists produced single platelet loss at concentrations that were similar to those previously found to cause aggregation of mouse platelets in traditional assays. 15,16 To validate our observations, we used a separate and distinct platelet marker, CD42d (GPV) and observed near-identical platelet counts when we analyzed separately for each marker over a range of activation conditions (r 2 5 0.998 by linear regression analysis; see supplemental Figure 1 on the Blood Web site).We next went on to confirm if single platelet loss in stimulated whole blood is an active, inhibitable process. To do this, we preincubated blood with the powerful platelet inhibitor PGI 2 and found significant reductions in the single platelet depletion induced by maximal tested concentrations of all agonists: collagen, 87 6 4% of single platelet loss vs 47 6 10% with PGI 2 ; PAR4-amide, 79 6 5% vs 25 6 9%; and U46619, 89 6 2% vs 26 6 7% ( Figure 2D-F).…”

mentioning

confidence: 81%

Novel whole blood assay for phenotyping platelet reactivity in mice identifies ICAM-1 as a mediator of platelet-monocyte interaction

Armstrong

Kirkby

Chan

et al. 2015

Blood

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Key Points• Low-volume, high-throughput whole blood aggregometry will facilitate future mouse platelet function research.• Application of this approach identifies ICAM-1 as a novel mediator of platelet-monocyte interaction through fibrinogen binding.Testing of platelet function is central to the cardiovascular phenotyping of genetically modified mice. Traditional platelet function tests have been developed primarily for testing human samples and the volumes required make them highly unsuitable for the testing of mouse platelets. This limits research in this area. To address this problem, we have developed a miniaturized whole blood aggregometry assay, based on a readily accessible 96-well plate format coupled with quantification of single platelet depletion by flow cytometric analysis. Using this approach, we observed a concentration-dependent loss of single platelets in blood exposed to arachidonic acid, collagen, U46619 or protease activated receptor 4 activating peptide. This loss was sensitive to well-established antiplatelet agents and genetic manipulation of platelet activation pathways. Observations were more deeply analyzed by flow cytometric imaging, confocal imaging, and measurement of platelet releasates. Phenotypic analysis of the reactivity of platelets taken from mice lacking intercellular adhesion molecule (ICAM)-1 identified a marked decrease in fibrinogen-dependent platelet-monocyte interactions, especially under inflammatory conditions. Such findings exemplify the value of screening platelet phenotypes of genetically modified mice and shed further light upon the roles and interactions of platelets in inflammation. (Blood. 2015;126(10):e11-e18) IntroductionPlatelets are key mediators of hemostasis. They have crucial roles in certain bleeding disorders and are the targets of antithrombotic therapies, notably aspirin and P2Y 12 receptor blockers such as clopidogrel. [1][2][3] Numerous assays have been developed to investigate platelet responses with an understandable focus on analyzing human clinical samples. 4,5 These assays require relatively large volumes of blood, with the majority requiring a sample volume in excess of 200 mL per test. 6 Mouse models are widely used in cardiovascular research, particularly for phenotyping of genetic modifications and testing novel therapeutic agents. However, platelet function testing in mice remains difficult because a complete and terminal blood collection provides at most 1 mL of blood. Further, commonly used strategies for extending the volume available such as dilution or platelet washing can introduce artifactual changes in platelet responses and thus does not permit study of platelet interactions with other blood cells and constituents. As a result, both the number and type of platelet tests that can be performed in samples from mice are severely limited, diminishing the experimental value of each mouse and making detailed study of mouse platelets impractical. There is therefore a clear and pressing experimental demand for murine-focused and optimized ...

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

Cited by 202 publications

References 39 publications

Stimulatory Roles of Nitric-oxide Synthase 3 and Guanylyl Cyclase in Platelet Activation

Stimulatory Roles of Nitric-oxide Synthase 3 and Guanylyl Cyclase in Platelet Activation

A Platelet Secretion Pathway Mediated by cGMP-dependent Protein Kinase

Novel whole blood assay for phenotyping platelet reactivity in mice identifies ICAM-1 as a mediator of platelet-monocyte interaction

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