Synergistic phosphorylation of platelet rap1B by SIN-1 and iloprost

Grünberg, Bernd; Negrescu, Emil V.; Siess, Wolfgang

doi:10.1016/0922-4106(95)90045-4

Cited by 12 publications

(11 citation statements)

References 19 publications

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“…4,5,35 This synergism is mainly caused by the inhibition of PDE III by cGMP, which enhances PGI 2 /cAMP signaling. 36,37 Accordingly, an increased concentration of cGMP due to PDE V inhibition by dipyridamole caused an enhanced synergism of cGK and cAK ( Figure 2). Interestingly, elevation of cGMP appears insufficient to increase basal cAMP levels and cAK effects (Figure 2).…”

Section: Aktas Et Al Dipyridamole and Platelet Inhibitionmentioning

confidence: 98%

Dipyridamole Enhances NO/cGMP-Mediated Vasodilator-Stimulated Phosphoprotein Phosphorylation and Signaling in Human Platelets

Aktas

Utz

Hoenig-Liedl

et al. 2003

Stroke

109

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Background and Purpose-Dipyridamole and in particular dipyridamole in combination with low-dose aspirin are very effective in preventing recurrent stroke. However, the mechanism(s) underlying this dipyridamole effect have not been elucidated. Since dipyridamole inhibits the cGMP-specific phosphodiesterase type V in vitro, we hypothesized and tested whether therapeutically relevant dipyridamole concentrations enhance NO/cGMP-mediated effects in intact human platelets studied ex vivo. Methods-Phosphorylation of vasodilator-stimulated phosphoprotein (VASP), an established marker of NO/cGMP effects in human platelets, was quantified by phosphorylation-specific antibodies and Western blots. Serotonin secretion and thromboxane synthase activity were determined by fluorometric quantification of derivatized serotonin and synthase products, respectively. Results-Endothelium-derived factors such as NO and prostaglandin I 2 are known to elevate both cGMP and cAMP levels with concomitant platelet inhibition and VASP phosphorylation. In our in vitro experiments, therapeutically relevant concentrations (3.5 mol/L) of dipyridamole amplified only cGMP-mediated VASP phosphorylation due to the NO donor sodium nitroprusside, but not cAMP-mediated effects. Furthermore, thromboxane synthase activity and serotonin secretion, events important for initial platelet activation, were inhibited by sodium nitroprusside, an effect also enhanced by dipyridamole, demonstrating the functional relevance of these observations. Finally, the ex vivo enhancement of NO/cGMP effects was also observed with platelets obtained from healthy volunteers treated with extended-release dipyridamole. Key Words: dipyridamole Ⅲ phosphodiesterase inhibitors Ⅲ platelet aggregation inhibitors Ⅲ stroke T he role of activated platelets in acute and chronic vascular diseases is well established. Accordingly, antiplatelet drugs significantly reduce the risk of severe events, such as ischemic stroke, myocardial infarction, and vascular death in atherosclerotic vascular disease. In numerous clinical studies, the efficacy of antiplatelet drugs in the treatment and secondary prevention of vascular diseases, particularly stroke, has been proven. [1][2][3] Platelet activation is inhibited by factors released from endothelial cells that constitute the inner cell layer of the vessel wall. The most important inhibitory endotheliumderived factors are NO and prostaglandin I 2 (PGI 2 ), which inhibit platelets by increasing the level of intracellular cyclic nucleotides. 4,5 Subsequent stimulation of cGMP-and cAMPdependent protein kinases leads to the phosphorylation of a variety of proteins. 6 In platelets, this inhibits agoniststimulated calcium signaling, 7,8 fibrinogen binding, 9 adhesion, 10 and aggregation 5 and stimulates phosphorylation of the cytoskeletal and focal adhesion protein vasodilatorstimulated phosphoprotein (VASP). 4,5,11 This phosphorylation correlates strongly with the inhibition of platelets. 9,11 Interestingly, antiplatelet treatment with clopidogrel or ticlo...

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Section: Aktas Et Al Dipyridamole and Platelet Inhibitionmentioning

confidence: 98%

Dipyridamole Enhances NO/cGMP-Mediated Vasodilator-Stimulated Phosphoprotein Phosphorylation and Signaling in Human Platelets

Aktas

Utz

Hoenig-Liedl

et al. 2003

Stroke

109

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“…The ability of NO to increase cAMP as well as that of PGI 2 to decrease cGMP levels indicates a crosstalk between cyclic nucleotides. The concept that in cells of megakaryocytic lineage, cAMP and cGMP downstream signaling events may interfere with each other was previously demonstrated in platelets (Maurice & Haslam, 1990; Grunberg et al ., 1995). These studies showed that NO‐mediated platelet inhibition involves not only cGMP but also cAMP increases due to PDE3A activity inhibition by cGMP.…”

Section: Discussionmentioning

confidence: 92%

Prostacyclin prevents nitric oxide‐induced megakaryocyte apoptosis

Pozner

Negrotto

D’Atri

et al. 2005

British J Pharmacology

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1 We have previously demonstrated that nitric oxide (NO) triggers CD34 þ -derived megakaryocyte apoptosis. We here show that prostacyclin (PGI 2 ) inhibits PAPA/NO-induced megakaryocyte death detected by fluorescent microscopy and flow cytometry. 2 The cAMP-specific phosphodiesterase inhibitor, Ro 20-1724, and the permeable analog dibutyrylcAMP also delayed apoptosis. PGI 2 effect was fully prevented when adenylyl cyclase activity was suppressed by SQ 22536, and partially reversed by the permeable protein kinase A inhibitor PKI 14-22 amide. ELISA showed that while both PGI 2 and NO alone or synergistically raised cAMP, only NO was able to increase intracellular cGMP levels. 3 Treatment of megakaryocytes with PGI 2 abolished both basal and NO-raised cGMP levels. Addition of 8-pCPT-cGMP or activation of soluble guanylyl cyclase by BAY 41-2272 induced cell death in a concentration-dependent manner, and ODQ, an inhibitor of guanylyl cyclase, prevented both PAPA/NO-or BAY 41-2272-induced apoptosis. Specific cGMP phosphodiesterase inhibition by Zaprinast or suppression of adenylyl cyclase by SQ 22536 enhanced the PAPA/NO proapoptotic effect. 4 PGI 2 completely inhibited NO-mediated generation and the increased activity of the cleaved form of caspase-3. 5 In conclusion, our results demonstrate that contrary to their well-known direct and synergistic inhibitory effects on platelets, PGI 2 and NO regulate opposite megakaryocyte survival responses through a delicate balance between intracellular cyclic nucleotide levels and caspase-3 activity control.

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“…When administered together, GEA 3175 and adenosine induced a synergistic inhibition of thrombin-stimulated platelet aggregation. A synergistic interaction between activators of cyclic GMP and cyclic AMP has previously been documented in platelets (Fisch et al, 1995;Grunberg et al, 1995). The underlying mechanism is thought to involve cyclic GMPinduced inhibition of class III phosphodiesterases (Maurice & Haslam, 1990).…”

Section: Discussionmentioning

confidence: 95%

“…However, the exact mechanisms underlying cyclic nucleotide-induced inhibition of platelet functions are still not completely understood. Furthermore, a combination of different compounds that increase either cyclic AMP or cyclic GMP has been shown to affect platelets in a synergistic manner (Maurice & Haslam 1990;Fisch et al, 1995;Grunberg et al, 1995). This indicates that intracellular cross-talk between ' Author for correspondence.…”

Section: Introductionmentioning

confidence: 99%

Synergistic inhibition of thrombin‐induced platelet aggregation by the novel nitric oxide‐donor GEA 3175 and adenosine

Grenegård

Gustafsson

Andersson

et al. 1996

British J Pharmacology

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The influence of the novel nitric oxide‐donor GEA 3175 on thrombin‐ and ionomycin‐stimulated human platelets was investigated. The effect of GEA 3175 was compared with that of adenosine, an activator of platelet adenylyl cyclase. GEA 3175 inhibited thrombin‐induced secretion of ATP but did not affect aggregation; similar results were obtained with adenosine. Thrombin‐stimulated rises in the cytosolic free Ca2+ concentration, [Ca2+]i, were dose‐dependently inhibited by GEA 3175 and adenosine. GEA 3175 and adenosine maximally reduced the initial rise in [Ca2+]i by 41% and 35%, respectively. Simultaneous exposure to GEA 3175 and adenosine nearly abolished both the functional responses (i.e. aggregation and degranulation) and the rises in [Ca2+]i in thrombin‐stimulated platelets. Aggregation and increases in [Ca2+]i triggered in platelets by the Ca2+‐ionophore ionomycin were only marginally affected by a combination of GEA 3175 and adenosine. GEA 3175 potently increased the guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) content in platelets but did not affect adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) levels. Adenosine did not increase either the cyclic AMP or the cyclic GMP levels in platelets. However, adenosine and GEA 3175 combined significantly elevated the platelet cyclic AMP content. The results show that simultaneous exposure to GEA 3175 and adenosine promotes potent anti‐aggregatory properties in platelets in vitro. The findings suggest that blockage of the cytosolic Ca2+‐signal, which is probably mediated by an amplified cyclic nucleotide response, is an important event during the synergistic inhibition of thrombin‐induced aggregation.

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Synergistic phosphorylation of platelet rap1B by SIN-1 and iloprost

Cited by 12 publications

References 19 publications

Dipyridamole Enhances NO/cGMP-Mediated Vasodilator-Stimulated Phosphoprotein Phosphorylation and Signaling in Human Platelets

Dipyridamole Enhances NO/cGMP-Mediated Vasodilator-Stimulated Phosphoprotein Phosphorylation and Signaling in Human Platelets

Prostacyclin prevents nitric oxide‐induced megakaryocyte apoptosis

Synergistic inhibition of thrombin‐induced platelet aggregation by the novel nitric oxide‐donor GEA 3175 and adenosine

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