Phosphorylation of rap1B by protein kinase a is not involved in platelet inhibition by cyclic AMP

Siess, Wolfgang; Grünberg, Bernd

doi:10.1016/0898-6568(93)90071-s

Cited by 18 publications

(11 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3335 Measurement of rap IB phosphorylation has been proposed as a biological marker for monitoring the actions of labile platelet inhibitors that elevate cAMP (prostacyclin, iloprost) and cGMP (nitric oxide, SIN-1) in platelets. 33 Stimulation of venous ECs with histamine, and in a similar experimental design incubation of pig coronary artery rings with bradykinin, elicited a reversible phosphorylation of VASP and a long-lasting phosphorylation of raplB. Inhibition of prostanoid synthesis by indomethacin and of EDRF formation by L-NMMA markedly reduced the phosphorylation of both platelet proteins, indicating that endothelium-derived prostacyclin and EDRF were biologically active in these experiments.…”

Section: Histamine Hlstamlnementioning

confidence: 85%

Formation of biologically active autacoids is regulated by calcium influx in endothelial cells.

Kruse¹,

Grünberg²,

Siess³

et al. 1994

Arterioscler Thromb

View full text Add to dashboard Cite

The blocker of receptor-mediated calcium entry SK&F 96365 was used to evaluate the contribution of calcium influx to the formation of biologically active endothelial prostanoids and endothelium-derived relaxing factor (EDRF). SK&F 96365 inhibited histamine-stimulated calcium entry into human umbilical vein endothelial cells but not its discharge from intracellular stores as determined spectrofluorometrically by changes of intracellular calcium concentration in fura-2-loaded cells. Concordantly, SK&F 96365 inhibited histamine-induced endothelial synthesis of 6-keto-prostaglandin F la and thromboxane B, in a dose-dependent manner. To assess the functional significance of endothelial formation of prostacyclin and EDRF to platelets, the cAMP-and cGMPdependent phosphorylation of two platelet proteins, raplB and a 50-kD vasodilator-stimulated phosphoprotein (VASP), was analyzed in coincubation experiments of endothelial cells with platelets. Autacoids released by histamine-stimulated E ndothelium-derived autacoids are crucial for the maintenance of local vascular homeostasis and endothelium-platelet interactions. Endothelial cells (ECs) respond to hormonal, chemical, and physical stimuli with the generation and release of prostacyclin and endothelium-derived relaxing factor (EDRF; nitric oxide or nitric oxide-releasing compounds), both of which are known to dilate vessels and inhibit platelet function in a paracrine manner. 15 Concomitantly, ECs also release the potent vasoconstrictor and stimulator of platelet aggregation thromboxane A 2 (TXA 2 ).6 ' 7The increase of cytosolic calcium concentration ([Ca 2+ ],), a key step in the stimulus-response coupling of ECs, leads to the activation of calcium-dependent enzymes such as phospholipase A 2 and nitric oxide synthase. 89 A variety of blood-borne mediators interact with endothelial surface receptors and raise [Ca 2+ ], by both inducing second messenger-mediated discharge of calcium from internal stores and promoting influx from the extracellular milieu.1 Calcium entry into nonexcitable cells occurs voltage independently via receptorcoupled channels of the plasma membrane that are not well characterized. 10Formation and release of EDRF and prostanoids by the vascular endothelium are coupled to an increase in Received May 31, 1994; accepted Jury 25, 1994. From the Institut fur Prophylaxe und Epidemiologie der Kreislaufkrankheiten, University of Munich, FRG.Correspondence to H.-J. Kruse, Institut fur Prophylaxe und Epidemiologie der Kreislaufkrankheiten, UniversitSt Munchen, Pettenkoferstr 9, 80336 Munchen, Germany.O 1994 American Heart Association, Inc.endothelial cells caused the phosphorylation of rap IB and VASP in platelets, which was only partly inhibited by either indomethacin or A r°-monomethyl-L-arginine but was almost completely suppressed by SK&F 96365. The concomitant endothelial release of thromboxane A 2 had no effect on protein kinase C-and calcium-dependent phosphorylation of platelet proteins. The results demonstrate that blockade of recepto...

show abstract

Section: Histamine Hlstamlnementioning

confidence: 85%

Formation of biologically active autacoids is regulated by calcium influx in endothelial cells.

Kruse¹,

Grünberg²,

Siess³

et al. 1994

Arterioscler Thromb

View full text Add to dashboard Cite

show abstract

“…Furthermore, the slow kinetics of Rap1 phosphorylation do not correlate with the fast inhibition of platelet activation by NO/cGMP (44). The cGKI substrate VASP has been shown to be involved in platelet adhesion (45), however, NO/cGMP effects on platelet aggregation were only marginally affected in VASP-deficient mice (46,47).…”

Section: Discussionmentioning

confidence: 99%

The NO/cGMP pathway inhibits Rap1 activation in human platelets via cGMP-dependent protein kinase I

Danielewski

Schultess²,

Smolenski³

2005

Thromb Haemost

View full text Add to dashboard Cite

SummaryThe NO/cGMP signalling pathway strongly inhibits agonist-induced platelet aggregation. However, the molecular mechanisms involved are not completely defined. We have studied NO/cGMP effects on the activity of Rap1, an abundant guanine-nucleotidebinding protein in platelets. Rap1-GTP levels were reduced by NO-donors and activators of NO-sensitive soluble guanylyl cyclase. Four lines of evidence suggest that NO/cGMP effects are mediated by cGMP-dependent protein kinase (cGKI): (i) Rap1 inhibition correlated with cGKI activity as measured by the phosphorylation state ofVASP, an established substrate of cGKI, (ii) 8-pCPT-cGMP, a membrane permeable cGMP-analog and activator of cGKI, completely blocked Rap1 activation, (iii) Rp- 8pCPT-cGMPS, a cGKI inhibitor, reversed NO effects and (iv) expression of cGKI in cGKI-deficient megakaryocytes inhibited Rap1 activation. NO/cGMP/cGKI effects were independent of the type of stimulus used for Rap1 activation. Thrombin-,ADPand collagen-induced formation of Rap1-GTP in platelets as well as turbulence-induced Rap1 activation in megakaryocytes were inhibited. Furthermore, cGKI inhibited ADP-induced Rap1 activation induced by the G α i -coupled P2Y12 receptor alone, i.e. independently of effects on Ca2+-signalling. From these studies we conclude that NO/cGMP inhibit Rap1 activation in human platelets and that this effect is mediated by cGKI. Since Rap1 controls the function of integrin α IIbβ 3 , we propose that Rap1 inhibition might play a central role in the anti-aggregatory actions of NO/cGMP.

show abstract

“…65 Early studies also showed direct phosphorylation of Rap1 by cGKI, but neither GTP-binding nor GTPase activity were influenced by phosphorylation, 66,67 and no correlation between Rap1 phosphorylation and inhibition of platelet activation could be detected. 68 Rap1GAP2a/b is a new effector of the NO/cGMP pathway, and we speculate that Rap1GAP2 phosphorylation by cGKI could enhance its GAP activity, thereby leading to Rap1 inactivation and ultimately to the inactivation of integrin ␣ IIb ␤ 3 . However, serine 7 phosphorylation does not appear to change the GAP activity of Rap1GAP2a/b directly.…”

Section: Phosphorylation Of Rap1gap2mentioning

confidence: 99%

Rap1GAP2 is a new GTPase-activating protein of Rap1 expressed in human platelets

Schultess

Danielewski

Smolenski

2005

Blood

View full text Add to dashboard Cite

The Ras-like guanine-nucleotide-binding protein Rap1 controls integrin ␣ IIb ␤ 3 activity and platelet aggregation. Recently, we have found that Rap1 activation can be blocked by the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) signaling pathway by type 1 cGMP-dependent protein kinase (cGKI). In search of possible targets of NO/cGMP/cGKI, we studied the expression of Rap1-specific GTPaseactivating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) in platelets. We could detect mRNAs for a new protein most closely related to Rap1GAP and for postsynaptic density-95 discslarge and zona occludens protein 1 (PDZ)-GEF1 and CalDAG-GEFs I and III. Using 5-rapid amplification of cDNA ends (RACE), we isolated the complete cDNA of the new GAP encoding a 715-amino acid protein, which we have termed Rap1GAP2. Rap1GAP2 is expressed in at least 3 splice variants, 2 of which are detectable in platelets. Endogenous Rap1GAP2 protein partially colocalizes with Rap1 in human platelets. In transfected cells, we show that Rap1GAP2 exhibits strong GTPase-stimulating activity toward Rap1. Rap1GAP2 is highly phosphorylated, and we have identified cGKI as a Rap1GAP2 kinase. cGKI phosphorylates Rap1GAP2 exclusively on serine 7, a residue present only in the platelet splice variants of Rap1GAP2. Phosphorylation of Rap1GAP2 by cGKI might mediate inhibitory effects of NO/cGMP on Rap1. IntroductionPlatelets are of great physiologic importance as regulators of clot formation and inflammation in the vasculature, and they have been established as major therapeutic targets in cardiovascular disease. 1 Platelets contain high levels of Rap1, a Ras-like guanine-nucleotidebinding protein. Recently, Rap1 was identified as a potent regulator of integrin function. [2][3][4][5] For example, the regulation of lymphocyte and macrophage adhesion by integrins ␣L␤ 2 and ␣B M2 , respectively, is controlled by Rap1. 6,7 Rap1 also facilitates the activation of platelet integrin ␣ IIb ␤ 3 , which is required for fibrinogen binding and aggregation. 8,9 Recently, Rap1 was shown to control the aggregation of mouse platelets. 10 Many different platelet agonists, including thrombin, adenosine diphosphate (ADP), collagen, and thromboxane, induce Rap1 activation. 11,12 However, the exact pathways involved in Rap1 activation in platelets are unknown. Data from other cell types suggest that receptor-mediated formation of second messengers can lead to the activation of Rap1-specific guanine nucleotide exchange factors (GEFs). Calcium and diacylglycerol activate CalDAG-GEFI (also termed RasGRP2), a protein detected in mouse megakaryocytes and platelets, 10,13 and CalDAG-GEFIII (also termed RasGRP3), involved in neuronal differentiation and B-cell development. 14,15 Cyclic adenosine monophosphate (cAMP) activates cAMP-dependent Epacs, 16,17 and C3G is regulated by tyrosine phosphorylation 18 and by binding to adaptor proteins. 19,20 Postsynaptic density-95 discs-large and zona occludens protein 1 (PDZ)-GEF1 is a ubiquitously expressed GEF of Rap1 and co...

show abstract

Phosphorylation of rap1B by protein kinase a is not involved in platelet inhibition by cyclic AMP

Cited by 18 publications

References 20 publications

Formation of biologically active autacoids is regulated by calcium influx in endothelial cells.

Formation of biologically active autacoids is regulated by calcium influx in endothelial cells.

The NO/cGMP pathway inhibits Rap1 activation in human platelets via cGMP-dependent protein kinase I

Rap1GAP2 is a new GTPase-activating protein of Rap1 expressed in human platelets

Contact Info

Product

Resources

About