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...

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...

A recently discovered phosphatidylinositol monophosphate, phosphatidylinositol 5-phosphate (PtdIns-5-P), plays an important role in nuclear signaling by influencing p53-dependent apoptosis. It interacts with a plant homeodomain finger of inhibitor of growth protein-2, causing an increase in the acetylation and stability of p53. Here we show that type I phosphatidylinositol-4,5-bisphosphate 4-phosphatase (type I 4-phosphatase), an enzyme that dephosphorylates phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2), forming PtdIns-5-P in vitro, can increase the cellular levels of PtdIns-5-P. When HeLa cells were treated with the DNAdamaging agents etoposide or doxorubicin, type I 4-phosphatase translocated to the nucleus and nuclear levels of PtdIns-5-P increased. This action resulted in increased p53 acetylation, which stabilized p53, leading to increased apoptosis. Overexpression of type I 4-phosphatase increased apoptosis, whereas RNAi of the enzyme diminished it. The half-life of p53 was shortened from 7 h to 1.8 h upon RNAi of type I 4-phosphatase. This enzyme therefore controls nuclear levels of PtdIns-5-P and thereby p53-dependent apoptosis.acetylated p53 ͉ inositol signaling ͉ nuclear translocation I nositol lipids participate in a variety of intracellular signaling pathways including cytoskeletal dynamics, intracellular membrane trafficking, cell proliferation, and apoptosis (1, 2). In response to agonists, the phosphoinositide profile is modulated by phospholipases, lipid kinases, and lipid phosphatases. The lipid messengers transduce signals through binding to proteins with binding domains specific for different phosphoinositides.The most recently discovered of the seven known phosphoinositides is phosphatidylinositol 5-phosphate (PtdIns-5-P), and its function is the least understood (3). The origin of PtdIns-5-P in cells was until recently unknown. A study of changes in the cellular levels of PtdIns-5-P suggested that PtdIns-5-P arises from the action of a phosphatase rather than a kinase (4). Our discovery of two phosphatases that convert PtdIns-4,5-P 2 to PtdIns-5-P provides a route for synthesis of this lipid (5). Recently, it was suggested that PtdIns-5-P specifically interacts with a plant homeodomain (PHD) finger of inhibitor of growth protein-2 (ING2) protein, and that this interaction is required for ING2-dependent activation of p53, which leads to increased apoptosis (6). This suggestion was based on the finding that RNAi of ING2 or overexpression of the phosphatidylinositol phosphate kinase (PIPK) type II␤, an enzyme that converts PtdIns-5-P to PtdIns-4,5-P 2 , decreases apoptosis. Thus, it was presumed that both ING2 and PtdIns-5-P were required for acetylation of p53, although cellular PtdIns-5-P was not measured in that study (6).The ING2 is a member of the inhibitor of growth family and acts as a cofactor on the histone acetyltransferase complex that functions in chromatin remodeling and p53 acetylation and activation (7). Mutation of the PHD finger that renders PtdIns-5-P-binding defective...

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.

Myotubularin-related protein 6 (MTMR6) is a catalytically active member of the myotubularin (MTM) family, which is composed of 14 proteins. Catalytically active myotubularins possess 3-phosphatase activity dephosphorylating phosphatidylinositol-3-phoshate and phosphatidylinositol-3,5-bisphosphate, and some members have been shown to form homomers or heteromeric complexes with catalytically inactive myotubularins. We demonstrate that human MTMR6 forms a heteromer with an enzymatically inactive member myotubularin-related protein 9 (MTMR9), both in vitro and in cells. MTMR9 increased the binding of MTMR6 to phospholipids without changing the lipid binding profile. MTMR9 increased the 3-phosphatase activity of MTMR6 up to 6-fold. We determined that MTMR6 is activated up to 28-fold in the presence of phosphatidylserine liposomes. Together, MTMR6 activity in the presence of MTMR9 and assayed in phosphatidylserine liposomes increased 84-fold. Moreover, the formation of this heteromer in cells resulted in increased protein levels of both MTMR6 and MTMR9, probably due to the inhibition of degradation of both proteins. Furthermore, co-expression of MTMR6 and MTMR9 decreased etoposide-induced apoptosis, whereas decreasing both MTMR6 and MTMR9 by RNA interference led to increased cell death in response to etoposide treatment when compared with that seen with RNA interference of MTMR6 alone. Thus, MTMR9 greatly enhances the functions of MTMR6.Myotubularin proteins are a family of 14 proteins with the canonical dual specificity protein tyrosine phosphatase active site CX 5 R motif (1-3). Eight members of the myotubularin family possess catalytic activity, dephosphorylating phosphatidylinositol 3-phosphate (PtdIns-3-P) 4 and phosphatidylinositol 3,5-bisphosphate (PtdIns-3,5-P 2 ) at the D-3 position, and six members are not catalytically active because they lack the conserved cysteine residue in the protein tyrosine phosphatase motif that is required for activity. Interest in this group of proteins originated from the genetic evidence linking myotubularin, the founding member of this family, to myotubular myopathy, an X-linked disorder characterized by severe hypotonia and generalized muscle weakness (4). Subsequently, mutations in MTMR2 and in its inactive binding partner MTMR13 were linked to a subset of Charcot-Marie-Tooth disease type 4B, a demyelinating neurodegenerative disorder (5, 6). Despite near identical substrate specificity, biochemical and genetic evidence supports the hypothesis that myotubularin proteins are not redundant and have unique functions within cells (2, 7-9). The mechanisms by which loss of function of myotubularin proteins produce diseases are not known. Current evidence supports the hypothesis that each myotubularin protein regulates a specific pool of PtdIns-3-P and/or PtdIns-3,5-P 2 , which in turn regulates a variety of cellular functions. Differences in tissue expression and subcellular localization play a role in the specificity of different myotubularins (10 -15).The functions of myotubulari...

The myotubularins are a large family of inositol polyphosphate 3-phosphatases that, despite having common substrates, subsume unique functions in cells that are disparate. The myotubularin family consists of 16 different proteins, 9 members of which possess catalytic activity, dephosphorylating phosphatidylinositol 3-phosphate [PtdIns(3)P] and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P 2 ] at the D-3 position. Seven members are inactive because they lack the conserved cysteine residue in the CX 5 R motif required for activity. We studied a subfamily of homologous myotubularins, including myotubularin-related protein 6 (MTMR6), MTMR7, and MTMR8, all of which dimerize with the catalytically inactive MTMR9. Complex formation between the active myotubularins and MTMR9 increases their catalytic activity and alters their substrate specificity, wherein the MTMR6/R9 complex prefers PtdIns(3,5)P 2 as substrate; the MTMR8/R9 complex prefers PtdIns(3)P. MTMR9 increased the enzymatic activity of MTMR6 toward PtdIns(3,5)P 2 by over 30-fold, and enhanced the activity toward PtdIns(3)P by only 2-fold. In contrast, MTMR9 increased the activity of MTMR8 by 1.4-fold and 4-fold toward PtdIns (3,5)P 2 and PtdIns(3)P, respectively. In cells, the MTMR6/R9 complex significantly increases the cellular levels of PtdIns(5)P, the product of PI(3,5)P 2 dephosphorylation, whereas the MTMR8/R9 complex reduces cellular PtdIns(3)P levels. Consequentially, the MTMR6/R9 complex serves to inhibit stress-induced apoptosis and the MTMR8/R9 complex inhibits autophagy.I nositol lipids play important roles in a variety of intracellular signaling pathways. In response to stimuli, the phosphoinositide profile is regulated by phospholipases, lipid kinases, and phosphatases. Understanding the roles of inositol signaling has expanded during the last decade and a number of these enzymes have been shown to cause diseases when mutated (1). The tumor-suppressor PTEN was discovered through positional cloning as being mutated in several types of cancer (2, 3). PTEN was subsequently shown to be a phosphatase, which dephosphorylates phosphatidylinositol 3,4,5-trisphosphate to generate phosphatidylinositol 4,5-bisphosphate, an activity that is lost in patients with PTEN mutations (4, 5). Mutations in the inositol polyphosphate 5-phosphatase OCRL cause the X-linked disorder Lowe syndrome, which is associated with mental retardation, blindness, and renal failure (6). Mutations in myotubularin cause myotubular myopathy (7), and mutations in myotubularin-related protein 2 (MTMR2) and MTMR13 cause a form of Charcot Marie Tooth disease type 4B, a demyelinating neurodegenerative disorder (8, 9).The myotubularin family consists of 16 different proteins, 9 members of which possess catalytic activity (10, 11) and 7 members that are inactive. Myotubularin proteins are not redundant and have unique functions within cells by regulating a specific pool of dephosphorylating phosphatidylinositol 3-phosphate [PtdIns(3)P] and phosphatidylinositol 3,5-bisphosphate [PtdIns (3...

Nitric oxide (NO) is a short lived secondary messenger, synthesized by nitric-oxide synthases (NOS). It is believed that the activity of inducible NOS (iNOS) is regulated primarily at the transcription level by inducing expression of iNOS mRNA and protein, which then continuously produces NO, until its degradation. Platelets do not have the nuclear transcriptional regulatory mechanisms of the iNOS gene and are believed to generate NO in response to agonist stimulation via endothelial NOS (eNOS). However, here we show that agonist-induced NO production is only partially eNOS-dependent and is also mediated by iNOS. Platelet agonist-induced NO production is significantly reduced in iNOS-knockout platelets. Platelet NO production occurs within seconds after agonist addition and is not accompanied by changes in iNOS protein levels, indicating a signaling-mediated functional activation mechanism of iNOS. Importantly, iNOS knock-out and iNOS inhibitors reduce agonist-induced platelet secretion and aggregation and cGMP levels, indicating that iNOS activation is important in stimulating platelets via the newly identified NO-cGMP-dependent platelet secretion pathway. Furthermore, iNOS knock-out mice have prolonged bleeding time, suggesting that this novel mode of regulation of iNOS activity plays a physiologically relevant role in hemostasis.

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