A Platelet Secretion Pathway Mediated by cGMP-dependent Protein Kinase

Li, Zhenyu; Zhang, Guoying; Marjanovic, Jasna; Ruan, Changgeng; Du, Xiaoping

doi:10.1074/jbc.m401532200

Cited by 67 publications

(59 citation statements)

References 51 publications

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“…2), indicating that the primary role for NOS3 is to stimulate aggregationdependent platelet secretion. This is consistent with our data on the role of PKG in platelet secretion, indicating NOS3 is upstream of cGMP elevation and PKG activation in stimulating platelet secretion (30). Interestingly, a previous study indicated a role for NOS3 in insulininduced secretion of ATP and other vasodilator compounds.…”

Section: Discussionsupporting

confidence: 93%

“…cGMP stimulates several intracellular molecules, including PKG, cGMP-gated ion channel, and cGMP-regulated phosphodiesterases. Although it was believed that cGMP inhibits platelet activation by activating PKG, we have shown that PKG in fact plays an important stimulatory role in platelet activation (28,30,36), as PKG knock-out mouse platelets and PKG inhibitor-treated human platelets showed significantly reduced platelet aggregation in response to low dose agonists including soluble agonists thrombin, thromboxane A2, and adhesive proteins VWF and collagen. On the other hand, the inhibitory effects of NO donors and cGMP analogs on human platelets were reversed by inhibitors of cAMPdependent protein kinase (37) and adenylyl cyclase (38), indicating that these effects require elevation of cAMP, possibly induced by inhibition of cGMP-regulated phosphodiesterase 3 (31).…”

Section: Discussionmentioning

confidence: 77%

“…Together, these data indicate a novel signaling pathway in which platelet agonists activate platelet NOS3, releasing NO, which in turn activates sGC, leading to elevation of intracellular cGMP. cGMP stimulates platelet secretion via a PKG-dependent pathway that induces the second wave of platelet aggregation (30).…”

Section: Nos3 Plays Stimulatory Roles In a Cgmp-dependentmentioning

confidence: 99%

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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: Discussionsupporting

confidence: 93%

Section: Discussionmentioning

confidence: 77%

Section: Nos3 Plays Stimulatory Roles In a Cgmp-dependentmentioning

confidence: 99%

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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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“…Recent investigations proposed the sequential activation of p38 and ERK pathways by PKG 7,8,21 as a novel "roadway" to ␣IIb␤3 activation. 22 These studies with human platelets again heavily relied on the use and functional effects of PKG inhibitors that (similar to p38 inhibitors) need to be used with caution and proper controls including nucleotides inactive with respect to PKG Figure 1.…”

Section: Resultsmentioning

confidence: 99%

Thrombin stimulation of p38 MAP kinase in human platelets is mediated by ADP and thromboxane A2 and inhibited by cGMP/cGMP-dependent protein kinase

Begonja

Geiger

Rukoyatkina

et al. 2006

Blood

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p38 MAP kinase in human platelets is activated by platelet agonists including thrombin, thromboxane A 2 (TxA 2 ), ADP, and others. However, both upstream mechanisms of p38 MAP kinase activation, and their downstream sequelae, are presently controversial and essentially unclear. Certain studies report sequential activation of cGMP-dependent protein kinase (PKG) and p38/ERK pathways by platelet agonists, leading to integrin activation and secretion, whereas others establish an essential role of Src/ERKmediated TxA 2 generation for fibrinogen receptor activation in human platelets. Here, we show that ADP secreted from platelet-dense granules, and subsequent activation of P2Y 12 receptors, as well as TxA 2 release are important upstream mediators of p38 MAP kinase activation by thrombin. However, p38 MAP kinase activation did not significantly contribute to calcium mobilization, P-selectin expression, ␣IIb␤3 integrin activation, and aggregation of human platelets in response to thrombin. Finally IntroductionIn vivo, circulating platelets are continually exposed to both adhesive and/or activating factors (fibrinogen, ADP, von Willebrand factor [VWF], thrombin, TxA 2 , etc), as well as inhibitory factors such as endothelium-derived nitric oxide (NO), prostacyclin (PG-I 2 ), and ADPase. 1 Most of these activating and inhibitory molecules bind to specific platelet receptors and stimulate signaling pathways that promote or inhibit platelet adhesion, aggregation, and secretion. A central role among platelet-activating factors is played by ADP, which induces multiple platelet responses and potentiates platelet aggregation caused by other agonists. 2,3 ADP is released from platelet-dense granules upon activation by agonists such as thrombin and collagen, and binds to purinergic receptors (P2Y 1 , P2Y 12 , P2X 1 ) to reinforce platelet aggregation and thrombus formation. 2,[4][5][6] Recently, new mechanisms for agonist-induced platelet activation and secretion were proposed to involve sequential activation of cGMP-dependent protein kinase (PKG)/p38/integrin or Akt/ eNOS/sGC/cGMP/PKG secretion, respectively, 7-9 whereas others indicate an essential role of Src/ERK-mediated TxA 2 generation for fibrinogen receptor activation in human platelets. 10 We 11,12 and others 10,13 were unable to reproduce the reported ERK activation by PKG. However, both the upstream mechanisms of p38 activation, and their downstream effects, are presently controversial and unclear. Here, we show that ADP secreted from platelet-dense granules, and subsequent activation of P2Y 12 receptors, as well as TxA 2 secretion are important mediators upstream of thrombin-evoked p38 activation. Furthermore, PKG activation does not stimulate, but rather inhibits, thrombin-evoked p38 activation, which alone has no significant effect on platelet stimulation/aggregation. Materials and methodsAnalysis of P-selectin expression, ␣IIb␤3 activation, aggregation, and intracellular calcium measurement Platelets were isolated from whole blood obtained from healthy volunteer...

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“…Data from a preliminary study of neutrophil degranulation suggest that release of enzymes from all three major classes of neutrophil granules is subjected to inhibition of PKG and PI3K (Table I). Recently, Li et al (52) reported that PKG plays an important role in aggregation-dependent release of dense granule and ␣-granule in platelets. Thus, PKG may be involved in degranulation triggered by multiple extracellular stimuli in different types of cells.…”

Section: Discussionmentioning

confidence: 99%

Regulation of Leukocyte Degranulation by cGMP-Dependent Protein Kinase and Phosphoinositide 3-Kinase: Potential Roles in Phosphorylation of Target Membrane SNARE Complex Proteins in Rat Mast Cells

Nanamori

Chen

et al. 2007

The Journal of Immunology

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We examined the roles of cGMP-dependent protein kinase (PKG) and PI3K in degranulation induced by fMLF and by FcεRI cross-linking. In rat basophilic leukemia-2H3 cells expressing formyl peptide receptor, the PKG inhibitors KT5823 and Rp-8-Br-PET-cGMP, as well as the PI3K inhibitor LY294002, reduced agonist-stimulated β-hexosaminidase release in a dose-dependent manner. These inhibitors also abolished vesicular fusion with the plasma membrane, as evidenced by diminished annexin V staining. Agonist-induced degranulation was completely blocked when LY294002 was applied together with one of the PKG inhibitors, suggesting an additive and possibly synergistic effect. In contrast, the PKG inhibitors did not affect fMLF-induced intracellular calcium mobilization and Akt phosphorylation. Likewise, LY294002 did not alter fMLF-induced elevation of intracellular cGMP concentration, and the inhibitory effect of LY294002 was not reversed by a cell-permeable analog of cGMP. Treatment with fMLF induced phosphorylation of soluble N-ethylmaleimide-sensitive factor-attachment protein (SNAP)-23, syntaxins 2, 4, and 6, and Monc18-3. The induced phosphorylation of SNAP-23 and syntaxins 2 and 4 was blocked by Rp-8-Br-PET-cGMP and LY294002. However, LY294002 was less effective in inhibiting Munc18-3 phosphorylation. The induced phosphorylation of syntaxin 6 was not effectively blocked by either Rp-8-Br-PET-cGMP or LY294002. Treatment of human neutrophils with the PKG inhibitors and LY294002 reduced enzyme release from primary, secondary, and tertiary granules. These results suggest that PKG and PI3K are involved in degranulation, possibly through phosphorylation of target membrane SNAP receptor proteins and their binding proteins.

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A Platelet Secretion Pathway Mediated by cGMP-dependent Protein Kinase

Cited by 67 publications

References 51 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

Thrombin stimulation of p38 MAP kinase in human platelets is mediated by ADP and thromboxane A2 and inhibited by cGMP/cGMP-dependent protein kinase

Regulation of Leukocyte Degranulation by cGMP-Dependent Protein Kinase and Phosphoinositide 3-Kinase: Potential Roles in Phosphorylation of Target Membrane SNARE Complex Proteins in Rat Mast Cells

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