Platelet rap1B Phosphorylation Is a Sensitive Marker for the Action of Cyclic AMP- and Cyclic GMP-Increasing Platelet Inhibitors and Vasodilators

Grünberg, Bernd; Kruse, Hans-Joachim; Negrescu, Emil V.; Siess, Wolfgang

doi:10.1097/00005344-199504000-00006

Cited by 16 publications

(11 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…phosphorylation of the PKA substrate, Rap1B, more effectively than the cell-permeable cGMP analogue, 8-pCPT-cGMP (Grü nberg et al, 1995).…”

Section: Discussionmentioning

confidence: 99%

“…NP is less effective in increasing cAMP only because of the simultaneous activation of PDE2 by cGMP (Dickinson et al , 1997). However, definitive evidence that the cAMP accumulating in response to NP or other NO donors can activate PKA is lacking, though SIN‐1 has been reported to stimulate the phosphorylation of the PKA substrate, Rap1B, more effectively than the cell‐permeable cGMP analogue, 8‐pCPT‐cGMP (Grünberg et al , 1995).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Roles for both cyclic GMP and cyclic AMP in the inhibition of collagen‐induced platelet aggregation by nitroprusside*

Jang¹,

Azzam²,

Dickinson

et al. 2002

Br J Haematol

View full text Add to dashboard Cite

Summary. In studies on human platelets, nitroprusside (NP) alone at 1-10 lmol/l increased platelet cyclic AMP (cAMP) by 40-70%, whereas increases in cyclic GMP (cGMP) were much larger in percentage though not in concentration terms. Collagen enhanced these increases in cAMP up to fourfold, without affecting cGMP. This effect was partly prevented by indomethacin or aspirin, indicating that platelet cyclo-oxygenase products acted synergistically with NP to increase cAMP. ADP released from the platelets by collagen tended to restrict this cAMP accumulation. Addition of 2¢,5¢-dideoxyadenosine (DDA), an inhibitor of adenylyl cyclase, decreased both the inhibition of collagen-induced platelet aggregation by NP and the associated accumulation of cAMP without affecting cGMP, indicating that cAMP mediates part of the inhibitory effect of NP. Unlike DDA, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of guanylyl cyclase, blocked all increases in both cGMP and cAMP caused by NP, as well as the inhibition of platelet aggregation, suggesting that cAMP accumulation was secondary to that of cGMP. Human platelet cGMP-dependent protein kinase (PKG) coelectrophoresed with the purified bovine type Ib isoenzyme. An inhibitor of this enzyme (R p )-b-phenyl-1,N 2 -etheno-8-bromoguanosine 3¢,5¢-cyclicmonophosphorothioate, diminished the inhibition of collagen-induced platelet aggregation by NP, but had little additional effect when DDA was present. This showed that both PKG and cAMP participate in the inhibition of collagen-induced platelet aggregation by NP. Moreover, selective activators of PKG and cAMP-dependent protein kinases had supra-additive inhibitory effects, suggesting that an optimal inhibitory effect of NP requires simultaneous activation of both enzymes.

show abstract

“…phosphorylation of the PKA substrate, Rap1B, more effectively than the cell-permeable cGMP analogue, 8-pCPT-cGMP (Grü nberg et al, 1995).…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Roles for both cyclic GMP and cyclic AMP in the inhibition of collagen‐induced platelet aggregation by nitroprusside*

Jang¹,

Azzam²,

Dickinson

et al. 2002

Br J Haematol

View full text Add to dashboard Cite

show abstract

“…However, Rap1B phosphorylation has a slow time course and does not affect GTPbinding or GTPase activity of Rap1 (25,27). In the present study we have investigated possible effects of NO on stimulus-induced Rap1 activation.…”

Section: Introductionmentioning

confidence: 96%

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

“…17 Phosphorylation of GP1b (24 kDa) and the thromboxane A 2 (TxA 2 ) receptor (48-53 kDa) may contribute to inhibition of platelet activation, 28 whereas the effects of phosphorylation of other peptides, such as rap1B, remains unknown. 29 The intracellular signaling effects caused by NO are not fully understood but involve inhibition of phospholipase C and inositol triphosphate (IP 3 )-mediated mobilization of [Ca ϩϩ ] i . 30,31 The current study investigates the effect of NO on the thrombininduced platelet shape change done in vitro by infusing solutions of NO into suspensions of aspirin-treated, gel-filtered human platelets in the presence of creatine phosphate/creatine phosphokinase (CP/CPK).…”

Section: Introductionmentioning

confidence: 99%

Protein kinase A mediates inhibition of the thrombin-induced platelet shape change by nitric oxide

Jensen

Selheim

Døskeland

et al. 2004

Blood

View full text Add to dashboard Cite

The thrombin-induced platelet shape change was blocked by nitric oxide (NO), as revealed by scanning electron microscopy, light transmission, and resistiveparticle volume determination. The inhibitory effect of NO was accompanied by an increase in levels of both cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) and phosphorylation of the vasodilator-stimulated phosphoprotein (VASP). However, the inhibition of the shape change was only mimicked by cAMP analogs (Sp-5,6-DClcBIMPS, 8-AHA-cAMP, and 8-CPT-cAMP) and not by cGMP analogs (8-Br-PETcGMP, 8-Br-cGMP, and 8-pCPT-cGMP). The effect of NO on the thrombin-induced shape change was prevented by the protein kinase A (PKA) antagonists Rp-8-BrcAMPS and Rp-cAMPS. The protein kinase G (PKG) antagonist Rp-8-CPTcGMPS strongly inhibited PKG-mediated 46-kDa VASP Ser239 phosphorylation, but did not inhibit the thrombin-induced shape change or the PKA-mediated VASP Ser157 phosphorylation. Whereas an inhibitor of cyclic nucleotide phosphodiesterase (PDE) 3A (milrinone) mimicked the effect of NO, inhibitors of PDE2 (erythro-9-(2-hydroxy-3-nonyl)adenine) and PDE5 (dipyridamole) were poorly effective. We concluded that (1) NO was a potent and reversible inhibitor of the platelet shape change, (2) the shape change was reversible, (3) the inhibitory effect of NO was mediated through activation of PKA, (4) the onset of the NO effect coincided with VASP Ser157 phosphorylation, and (5) removal of NO and platelet shape change coincided with VASP Ser157 dephosphorylation. These findings are compatible with elevation of cGMP by NO in a compartment close to PDE3A, PKA, and VASP, leading to a local increase of cAMP able to block thrombin-induced shape change. IntroductionWhen platelets are stimulated, they rapidly change shape from discoids to spheres possessing extensive pseudopodia. [1][2][3] This shape change precedes aggregation, secretion, and activation of the coagulation cascade. 1,2,4 In healthy individuals, 65% to 90% of circulating platelets are discoidal, 2 and increased numbers of spherical platelets have been associated with vascular diseases. 5 Shape change could therefore be an important step in hemostasis and thrombosis. Activation of the platelet shape change by thrombin has been extensively studied in vitro, but little is known about mechanisms inhibiting this process. Thrombin, a potent platelet agonist that induces shape change, aggregation, and secretion through many signaling systems 6 is formed in vivo, in significant amounts at the site of a vascular injury, stimulating both platelet activation and overall thrombus growth. [7][8][9][10][11][12] Regulation of platelet activation is an important physiologic function of the vascular endothelium including release of nitric oxide (NO), 13 a potent inhibitor of platelet aggregation, secretion, adhesion, and binding of fibrinogen to integrin glycoprotein IIbIIIa (GpIIbIIIa). [14][15][16][17] Conflicting results have, however, been reported about the effect of NO on shape change. [18][19][20][...

show abstract

Platelet rap1B Phosphorylation Is a Sensitive Marker for the Action of Cyclic AMP- and Cyclic GMP-Increasing Platelet Inhibitors and Vasodilators

Cited by 16 publications

References 0 publications

Roles for both cyclic GMP and cyclic AMP in the inhibition of collagen‐induced platelet aggregation by nitroprusside*

Roles for both cyclic GMP and cyclic AMP in the inhibition of collagen‐induced platelet aggregation by nitroprusside*

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

Protein kinase A mediates inhibition of the thrombin-induced platelet shape change by nitric oxide

Contact Info

Product

Resources

About