Taming platelets with cyclic nucleotides

doi:10.1016/S0006-2952(01)00760-2

Biochemical Pharmacology

Volume 62, Issue 9, 1 November 2001, Pages 1153-1161

https://doi.org/10.1016/S0006-2952(01)00760-2 Get rights and content

Abstract

Cardiovascular diseases are often accompanied and aggravated by pathologic platelet activation. Tight regulation of platelet function is an essential prerequisite for intact vessel physiology or effective cardiovascular therapy. Physiological platelet antagonists as well as various pharmacological vasodilators inhibit platelet function by activating adenylyl and guanylyl cyclases and increasing intracellular cyclic AMP (cAMP) and cyclic GMP (cGMP) levels, respectively. Elevation of platelet cyclic nucleotides interferes with basically all known platelet activatory signaling pathways, and effectively blocks complex intracellular signaling networks, cytoskeletal rearrangements, fibrinogen receptor activation, degranulation, and expression of pro-inflammatory signaling molecules. The major target molecules of cyclic nucleotides in platelets are cyclic nucleotide-dependent protein kinases that mediate their effects through phosphorylation of specific substrates. They directly affect receptor/G-protein activation and interfere with a variety of signal transduction pathways, including the phospholipase C, protein kinase C, and mitogen-activated protein kinase pathways. Regulation of these pathways blocks several steps of cytosolic Ca²⁺ elevation and controls a multitude of cytoskeleton-associated proteins that are directly involved in organization of the platelet cytoskeleton. Due to their multiple sites of action and strong inhibitory potencies, cyclic nucleotides and their regulatory pathways are of particular interest for developing new approaches for the treatment of thrombotic and cardiovascular disorders.

Introduction

Platelets, platelet-vessel wall interactions, and platelet-derived factors are essential for the regulation of vascular tone and hemostasis. Platelets are activated by a number of stimuli resulting in platelet shape change, expression and/or activation of surface receptors, secretion of vasoactive substances, adhesion, aggregation, and finally thrombus formation. Vasoconstrictors (TxA₂, vasopressin) and endothelium-dependent vasodilators (ADP, thrombin, serotonin) are potent physiologic platelet activators. The initial event in platelet activation is binding of such agonists to specific membrane-spanning G-protein-linked receptors on the platelet surface, which transmit the signal via heterotrimeric and small GTP-binding proteins and multiple protein kinases into an ordered cascade of intracellular signaling pathways. All these pathways induce remodeling of the platelet cytoskeleton resulting in the appearance of a variety of F-actin structures, including filopodia, lamellipodia, and adhesion plaques. Platelet activation is tightly regulated under physiological conditions; however, it is often impaired in cardiovascular diseases and contributes to the development and aggravation of atherosclerosis, ischemic heart disease, diabetes, and other thrombotic disorders. Physiological platelet antagonists, like the endothelium-derived vasodilators PG-I₂ and EDRF, inhibit platelet function by activation of ACs and GCs, and increase intracellular levels of the cyclic nucleotides cAMP and cGMP, respectively. Also, various pharmacological vasodilators, like PG-E₁, sodium nitroprusside, and organic nitrates, use the potency of cyclic nucleotides to inhibit platelet function.

This review will focus on the regulation of cyclic nucleotide levels in human platelets and the known as well as potential target sites within platelet activatory signaling pathways that are inhibited by cyclic nucleotide-regulated signaling molecules.

Section snippets

Cyclic nucleotide levels in human platelets

Elevation of intracellular cAMP and cGMP is the most potent endogenous mechanism of platelet inhibition. Cyclic nucleotide levels are up-regulated by synthesis through ACs and GCs and down-regulated by degradation through PDEs. Major target enzymes of cyclic nucleotides involve cAMP-PK and cGMP-PK, which mediate their effects through phosphorylation of specific substrate proteins.

Regulation of AC and GC activity

ACs are integral membrane glycoproteins that catalyze the synthesis of cAMP from ATP, leading to an increased level of intracellular cAMP. Platelet AC is activated by the α subunit of the stimulatory G-protein (Gα_s), and strongly inhibited by the α subunit of the pertussis toxin-sensitive inhibitory G-protein (Gα_i) (Fig. 1) (reviewed in Ref. [1]). Binding of prostaglandins (PG-I₂, PG-E₁, PG-E₂) to their receptor, which is coupled to G_s, therefore leads to stimulation of cAMP formation.

PDEs

PDEs are a large group of enzymes consisting of several isozyme families that hydrolyze the 3′-phosphoester bond on cAMP and/or cGMP, converting them into biologically inactive 5′-nucleotide metabolites. Platelets contain at least three different types of PDEs (Table 1 and Fig. 1): the cGMP-stimulated PDE2, the cGMP-inhibited PDE3, and the cGMP-binding cGMP-specific PDE5 (reviewed in Ref. 6). PDE2 hydrolyzes both cAMP and cGMP, with similar affinities (K_m values of 50 and 35 μM, respectively),

Effector sites of cAMP and cGMP

Activation of cAMP- and cGMP-dependent protein kinases is the most important mechanism of cAMP and cGMP action in human platelets, as demonstrated by studies with membrane-permeable cyclic nucleotide analogs that selectively activate either cAMP-PK or cGMP-PK and do not affect PDEs. Inhibition of platelet aggregation by these analogs correlated well with activation of cAMP-PK or cGMP-PK in intact platelets [13], [14]. Furthermore, in contrast to platelets from healthy persons, cGMP-PK-deficient

Cyclic nucleotide-dependent protein kinases, their substrates and functional roles in platelets

Cyclic nucleotide-dependent protein kinases are the major effector molecules mediating physiological effects initiated by cyclic nucleotide formation. Compared with other tissues and cell types, human platelets contain particularly high concentrations of both cAMP-PK and cGMP-PK [17], pointing to the functional importance of protein phosphorylation in response to cyclic nucleotide-elevating platelet inhibitors. cAMP-PK types I and II β and cGMP-PK type I β represent the major forms of

Conclusion

Inhibition of platelet function by many clinically used platelet antagonists blocks only certain functions of platelet activation, leaving others untouched. For example, selective blockade of fibrinogen binding to activated platelets will not prevent expression of pro-inflammatory molecules. The increase of intracellular cyclic nucleotide levels leads to broad inhibition of platelet functions, including adhesion, degranulation, aggregation, and even down-regulation of pro-inflammatory platelet

Acknowledgements

We thank Dr. E. Butt and Dr. S.M. Lohmann for helpful suggestions.

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¹: Abbreviations: ABP, actin binding protein; AC, adenylyl cyclase; cAMP, cyclic AMP; cAMP-PK, cAMP-dependent protein kinase; cGMP, cyclic GMP; cGMP-PK, cGMP-dependent protein kinase; DAG, 1,2-diacylglycerol; EDRF, endothelium-derived relaxing factor; GC, guanylyl cyclase; GP, glycoprotein; Hsp27, heat shock protein 27; IP₃, inositol 1,4,5- trisphosphate; IRAG, IP₃ receptor-associated cGMP-PK substrate; MAPK, mitogen-activated protein kinase; MAPKAPK-2, MAPK-activated protein kinase-2; MARCKS, myristoylated alanine-rich C kinase substrate; MLC, myosin light chain; MLCK, myosin light chain kinase; PDE, phosphodiesterase; PG-E1, prostaglandin E₁; PG-I₂, prostacyclin; PIP₂, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; PLC, phospholipase C; TxA₂, thromboxane A₂, and VASP, vasodilator-stimulated phosphoprotein.

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Taming platelets with cyclic nucleotides1

Abstract

Introduction

Section snippets

Cyclic nucleotide levels in human platelets

Regulation of AC and GC activity

PDEs

Effector sites of cAMP and cGMP

Cyclic nucleotide-dependent protein kinases, their substrates and functional roles in platelets

Conclusion

Acknowledgements

Eur J Pharmacol

J Biol Chem

Atherosclerosis

Trends Pharmacol Sci

Biochem Pharmacol

J Biol Chem

Biochim Biophys Acta

J Biol Chem

Blood

J Biol Chem

J Biol Chem

Trends Biochem Sci

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

Biochem Biophys Res Commun

J Biol Chem

J Biol Chem

J Biol Chem

Curr Opin Cell Biol

J Biol Chem

J Biol Chem

Biochem Biophys Res Commun

Biochem Pharmacol

Blood

Blood

J Biol Chem

J Biol Chem

Biochem Biophys Res Commun

Taming platelets with cyclic nucleotides¹