Cyclic nucleotide elevating vasodilators inhibit platelet aggregation at an early step of the activation cascade

doi:10.1016/0014-2999(89)90165-9

European Journal of Pharmacology

Volume 159, Issue 3, 17 January 1989, Pages 317-320

https://doi.org/10.1016/0014-2999(89)90165-9 Get rights and content

Abstract

The mechanism of vasodilator-induced inhibition of platelet aggregation was investigated in human platelets. Cyclic nucleotide-elevating vasodilators stimulated cAMP- or cGMP-dependent protein phosphorylation, inhibited the activation of both protein kinase C and myosin light chain kinase, and inhibited the thrombin-induced hydrolysis of phosphatidylinositol-4,5-bisphosphate without affecting its resynthesis. The results suggest that cAMP- and cGMP-elevating vasodilators both inhibit platelet aggregation at an early step of the activation cascade, presumably at the level of phospholipase C.

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2019, Pharmacology and Therapeutics
For many millions of patients at secondary risk of coronary thrombosis pharmaceutical protection is supplied by dual anti-platelet therapy. Despite substantial therapeutic developments over the last decade recurrent thrombotic events occur, highlighting the need for further optimisation of therapies. Importantly, but often ignored, anti-platelet drugs interact with cyclic nucleotide systems in platelets and these are the same systems that mediate key endogenous pathways of platelet regulation, notably those dependent upon the vascular endothelium. The aim of this review is to highlight interactions between the anti-platelet drugs, aspirin and P2Y₁₂ receptor antagonists and endogenous pathways of platelet regulation at the level of cyclic nucleotides. These considerations are key to concepts such as anti-platelet drug resistance and individualized anti-platelet therapy which cannot be understood by study of platelets in isolation from the circulatory environment. We also explore novel and emerging therapies that focus on preserving haemostasis and how the concepts outlined in this review could be exploited therapeutically to improve anti-thrombotic efficacy whilst reducing bleeding risk.
Effects of the NO/soluble guanylate cyclase/cGMP system on the functions of human platelets
2018, Nitric Oxide - Biology and Chemistry
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Conversely, two counteracting systems, including sarcoplasmic/endoplasmic Ca2+ ATPases (SERCAs), which pump Ca2+ back into intracellular stores, and plasma membrane Ca2+ ATPases (PMCAs) which pump it out of cells, have been identified in platelets [104]. It has been known for a long time that cGMP-elevating agents inhibit platelet agonist (thrombin, TXA2)-activation of phospholipase Cß, generation of IP3 and 1,2 diacylglycerol, and subsequent PKC activation and rise of [iCa2+] [19,105,106], although the mechanisms remained unclear for many years. It now seems likely that cGMP-elevating agents inhibit platelet action at several sites, as also suggested by our ongoing phosphoproteomic studies.
Platelets are circulating sentinels of vascular integrity and are activated, inhibited, or modulated by multiple hormones, vasoactive substances or drugs. Endothelium- or drug-derived NO strongly inhibits platelet activation via activation of the soluble guanylate cyclase (sGC) and cGMP elevation, often in synergy with cAMP-elevation by prostacyclin. However, the molecular mechanisms and diversity of cGMP effects in platelets are poorly understood and sometimes controversial. Recently, we established the quantitative human platelet proteome, the iloprost/prostacyclin/cAMP/protein kinase A (PKA)-regulated phosphoproteome, and the interactions of the ADP- and iloprost/prostacyclin-affected phosphoproteome. We also showed that the sGC stimulator riociguat is in vitro a highly specific inhibitor, via cGMP, of various functions of human platelets. Here, we review the regulatory role of the cGMP/protein kinase G (PKG) system in human platelet function, and our current approaches to establish and analyze the phosphoproteome after selective stimulation of the sGC/cGMP pathway by NO donors and riociguat. Present data indicate an extensive and diverse NO/riociguat/cGMP phosphoproteome, which has to be compared with the cAMP phosphoproteome. In particular, sGC/cGMP-regulated phosphorylation of many membrane proteins, G-proteins and their regulators, signaling molecules, protein kinases, and proteins involved in Ca²⁺ regulation, suggests that the sGC/cGMP system targets multiple signaling networks rather than a limited number of PKG substrate proteins.
Phosphorylation and Regulation of G-protein-activated Phospholipase C-β3 by cGMP-dependent Protein Kinases
2001, Journal of Biological Chemistry
Citation Excerpt :
NO inhibits IP3 generation and intracellular Ca2+ mobilization by increasing the intracellular cGMP concentration. The downstream target for cGMP is believed to be the PKGs, which mediate the effects of cGMP in a number of cells or tissues, including the vascular smooth muscle and platelets (9, 31, 40, 51, 53). Whereas an increase in cGMP level correlates well with a decrease in intracellular Ca2+ concentration and the relaxation of the vascular smooth muscle in a time- and concentration-dependent manner, the molecular mechanisms for cGMP-PKG to regulate intracellular Ca2+ signaling are not well defined.
Among the drugs that are known to relax the vascular smooth muscle and regulate other cellular functions, β-adrenergic agonists and nitric oxide-containing compounds are some of the most effective ones. The mechanisms of these drugs are thought to lower agonist-induced intracellular [Ca²⁺] by increasing intracellular cAMP and cGMP, activating their respective protein kinases. However, the physiological targets of cyclic nucleotide-dependent protein kinases are not clear. The molecular basis for the regulation of intracellular Ca²⁺ by signaling pathways coupled to cyclic nucleotides is not well defined. G-protein-activated phospholipase C (PLC-β) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphates to generate diacylglycerol and inositol 1,4,5-triphosphate, leading to the activation of protein kinase C and the mobilization of intracellular Ca²⁺. In this study, we shown that G-protein-activated PLC enzymes are the potential targets of cGMP-dependent protein kinases (PKG). PKG can directly phosphorylate PLC-β2 and PLC-β3 in vitro with purified proteins and in vivo with metabolic labeling. Phosphorylation of PLC-β leads to the inhibition of G-protein-activated PLC-β3 activity by 50–70% in COS-7 cell transfection assays. By using phosphopeptide mapping and site-directed mutagenesis, we further identified two key phosphorylation sites for the regulation of PLC-β3 by PKG (Ser²⁶ and Ser¹¹⁰⁵). Mutation at these two sites (S26A and S1105A) of PLC-β3 completely blocked the phosphorylation of PLC-β3 protein catalyzed by PKG. Furthermore, mutation of these serine residues removed the inhibitory effect of PKG on the activation of the mutant PLC-β3 proteins by G-protein subunits. Our results suggest a molecular mechanism for the regulation of G-protein-mediated intracellular [Ca²⁺] by the NO-cGMP-dependent signaling pathway.
Inhibition of agonist-induced p42 and p38 mitogen-activated protein kinase phosphorylation and CD40 ligand/P-selectin expression by cyclic nucleotide-regulated pathways in human platelets
2000, Biochemical Pharmacology
Citation Excerpt :
However, the target substrates of cAMP-PK and cGMP-PK which initiate inhibition of both platelet granule secretion and MAPK activation remain to be identified. Earlier, it was shown that cyclic nucleotides inhibit platelet aggregation at an early step of the activation cascade, presumably at the level of phospholipase C [44]. This supports the hypothesis that platelet degranulation is affected at a level proximal to PKC activation [26, 27].
Platelet activation and adhesion to endothelial cells and extracellular matrix proteins are crucial events in the development of arterial cardiovascular diseases. Platelet activation is initiated by stimulation of intracellular signaling cascades, including the p42 mitogen-activated protein kinase (MAPK) and p38 MAPK pathways, followed by major changes in the platelet cytoskeleton and expression and activation of platelet surface receptors, such as P-selectin (CD62P) and CD40 ligand (CD40L). Activated platelets directly bind to vascular endothelial cells via CD40L/CD40 interactions and induce inflammatory reactions that initiate or aggravate atherosclerotic lesions. The aim of this study was to investigate effects of two known platelet inhibitors—the cAMP-elevating prostaglandin E₁ (PG-E₁) and the cGMP-elevating sodium nitroprusside (SNP)—on platelet p42 MAPK and p38 MAPK activation as well as on surface expression of CD62P and CD40L. MAPK activation was analyzed by Western blot experiments using phosphorylation-specific antibodies, and surface CD40L and CD62P expression was determined by flow cytometry analysis. PG-E₁ and SNP strongly inhibited p42 and p38 MAPK phosphorylation as well as CD40L and CD62P expression in response to thrombin, a thromboxane A₂ analog, and ADP. These data indicate that adenosine and guanosine 3′,5′-cyclic monophosphate-dependent protein kinases not only inhibit platelet pathways leading to activation and aggregation, but also those resulting in enhanced surface expression of protein ligands involved in inflammation. Expression of CD40L and CD62P was found to be independent of MAPK activation, since it was not inhibited by specific MAPK inhibitors. Inhibition of platelet-induced inflammatory responses including CD62P- and CD40L-mediated interaction of platelets with leukocytes and endothelial cells, respectively, is suggested to be an important component of the long-term vasoprotective effects of cyclic nucleotide-elevating prostaglandins and NO donors.
Regulation of human endothelial cell focal adhesion sites and migration by cGMP-dependent protein kinase I
2000, Journal of Biological Chemistry
Citation Excerpt :
However, VASP phosphorylation may be only one of the effects of cGK I on the cytoskeletal system. cGK I in platelets is known to inhibit phospholipase C and myosin light chain (MLC) phosphorylation (10, 55), which are suggested to be involved in tension- and stress fiber-mediated focal adhesion assembly (56, 57). Inhibition of MLC phosphorylation is unlikely to be mediated by cGK I phosphorylation of MLC kinase since this did not affect MLC kinase activity (58).
cGMP-dependent protein kinase type I (cGK I), a major constituent of the atrial natriuretic peptide (ANP)/nitric oxide/cGMP signal transduction pathway, phosphorylates the vasodilator-stimulated phosphoprotein (VASP), a member of the Ena/VASP family of proteins involved in regulation of the actin cytoskeleton. Here we demonstrate that stimulation of human umbilical vein endothelial cells (HUVECs) by both ANP and 8-(4-chlorophenylthio)guanosine 3′:5′-monophosphate (8-pCPT-cGMP) activates transfected cGK I and causes detachment of VASP and its known binding partner (zyxin) from focal adhesions in >60% of cells after 30 min. The ANP effects, but not the 8-pCPT-cGMP effects, reversed after 3 h of treatment. In contrast, a catalytically inactive cGK Iβ mutant (cGK Iβ-K405A) was incapable of mediating these effects. VASP mutated (Ser/Thr to Ala) at all three of its established phosphorylation sites (vesicular stomatitis virus-tagged VASP-AAA mutant) was not phosphorylated by cGK I and was resistant to detaching from HUVEC focal adhesions in response to 8-pCPT-cGMP. Furthermore, activation of cGK I, but not of mutant cGK Iβ-K405A, caused a 1.5–2-fold inhibition of HUVEC migration, a dynamic process highly dependent on focal adhesion formation and disassembly. These results indicate that cGK I phosphorylation of VASP results in loss of VASP and zyxin from focal adhesions, a response that could contribute to cGK alteration of cytoskeleton-regulated processes such as cell migration.
Stimulation of calcium influx and platelet activation by canatoxin: Methoxyverapamil inhibition and downregulation by cGMP
1997, Archives of Biochemistry and Biophysics
Canatoxin (CNTX), a toxic protein isolated from seeds ofCanavalia ensiformis,induces Ca²⁺influx across the platelet plasma membrane, mobilization of arachidonic acid mediated by phospholipase A₂, ATP secretion, and platelet aggregation. All these events depend on the presence of extracellular Ca²⁺and are blocked by methoxyverapamil, a calcium-channel blocker. CNTX does not activate phospholipase C, and the intracellular calcium mobilization mediated by IP₃does not play a role in platelet activation by this toxin. Preincubation of rabbit platelets with 8-Br-cGMP inhibited the CNTX-evoked calcium influx, arachidonate release, ATP secretion, and cell aggregation. Our data suggest that the calcium influx is a prior step on platelet activation by CNTX, being modulated by cGMP.

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¹: Present address: Department of Pharmacology, Stanford University School of Medicine, Palo Alto, CA 94304, U.S.A.

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Short communicationCyclic nucleotide elevating vasodilators inhibit platelet aggregation at an early step of the activation cascade

Abstract

J. Biol. Chem.

J. Biol. Chem.

Trends Pharmacol. Sci.

J. Biol. Chem.

Short communication
Cyclic nucleotide elevating vasodilators inhibit platelet aggregation at an early step of the activation cascade