Phosphoinositide 3-Kinase Inhibition Spares Actin Assembly in Activating Platelets but Reverses Platelet Aggregation

The ability of leukocytes to self-regulate adhesion during transendothelial and extravascular migration is fundamental to the performance of immune surveillance in complex extracellular matrices. Leukocyte adhesion is regulated through the modulation of integrin receptors such as ␣ v ␤ 3 . In this study, we examined the activation of ␣ v ␤ 3 resulting from attachment to vitronectin or fibronectin. In K562 cells stably expressing transfected ␣ v ␤ 3 , adhesion to vitronectin required tyrosine phosphorylation of the ␤ 3 subunit and activation of phosphoinositide 3-kinase and protein kinase C. In contrast, adhesion to fibronectin proceeded without ␤ 3 -tyrosine phosphorylation or the activities of phosphoinositide 3-kinase or protein kinase C. Firm adhesion to both ligands and actin stress fiber formation required both Syk and Rho activity, suggesting that each ligand employs unique signaling pathways to achieve an active integrin complex, likely merging at a common RhoGEF such as Vav. Distinct signaling by a single integrin species interacting with different ligands permits initiation of additional cellular processes specific to the current task and provides an explanation for what has been described as promiscuous ligand specificity among integrins.

Section: Discussioncontrasting

confidence: 55%

Ligand-dependent Activation of Integrin αvβ3

Butler

Williams

Blystone

2003

“…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: 43%

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

Marjanovic

Stojanović

et al. 2005

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...

“…Free 32 P was separated from platelets by gel filtration over a Sepharose 2B column in the presence of 1 M prostaglandin E 1 . Following activation of platelets with thrombin or PMA with stirring in the presence of 500 g/ml fibrinogen, lipids were extracted in chloroform:methanol:HCl, deacylated, and analyzed by high pressure liquid chromatography as previously described (12,13).…”

Section: Methodsmentioning

confidence: 99%

D3 Phosphoinositides and Outside-in integrin Signaling by Glycoprotein IIb-IIIa Mediate Platelet Actin Assembly and Filopodial Extension Induced by Phorbol 12-Myristate 13-Acetate

Hartwig

Kung

Kovacsovics

et al. 1996

Self Cite

126

109

Phorbol 12-myristate 13-acetate (PMA) uncaps a small number of the fast-growing (barbed) ends of actin filaments, thereby eliciting slow actin assembly and extension of filopodia in human blood platelets. These reactions, which also occur in response to immunologic perturbation of the integrin glycoprotein (GP) IIb-IIIa, are sensitive to the phosphoinositide 3-kinase inhibitor wortmannin. Platelets deficient in GPIIb-IIIa integrins or with GPIIb-IIIa function inhibited by calcium chelation or the peptide RGDS have diminished PMA responsiveness. The effects of PMA contrast with thrombin receptor stimulation by 5 M thrombin receptor-activating peptide (TRAP), which causes rapid and massive wortmannin-insensitive actin assembly and lamellar and filopodial extension. However, we show here that wortmannin can inhibit filopod formation if the thrombin receptor is ligated using suboptimal doses (<1 M) of TRAP. Phosphatidylinositol 3,4-bisphosphate inhibits actin filament severing and capping by human gelsolin in vitro. The findings implicate D3 polyphosphoinositides and integrin signaling in PMA-mediated platelet stimulation and implicate D3 containing phosphoinositides generated in response to protein kinase C activation and GPIIb-IIIa signaling as late-acting intermediates leading to filopodial actin assembly.Extracellular perturbations cause cells to change cell shape and translocate by remodeling diverse intracellular actin-based structures. The relative functional simplicity of these changes in the anucleate human blood platelet makes it a useful subject for working out the pathways leading to this remodeling. Of particular utility is the fact that actin remodeling events in the platelet predominantly take place sequentially in time rather than the actin rearrangements that occur simultaneously in space during cell locomotion.The resting platelet is a rigid disc stabilized by an actin filament gel that links to a submembrane skeleton and to transmembrane proteins (1, 2). Over 98% of the actin filaments in resting platelets have their fast growing ("barbed" as defined by myosin head fragment binding) ends stabilized by specific barbed-end capping proteins. In response to thrombin, a calcium transient in the platelet activates gelsolin, which then severs actin filaments in the periphery of the actin filament gel and caps the newly formed barbed ends of the severed filaments (3). Subsequently, phosphoinositides accumulate and inactivate gelsolin as well as another abundant barbed end binding factor, capping protein. Phosphoinositide-mediated uncapping of at least 25% of the filament barbed ends eventuates in massive actin assembly as micromolar quantities of monomeric subunits, previously prevented from spontaneous nucleation by sequestering proteins, add onto the uncapped filament barbed ends (4 -6).The actin assembly following thrombin-induced platelet activation mediates the appearance of two types of surface protrusions, ruffling lamellae that spread circumferentially and threadlike filopodial protrusions. The fi...