|
 
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| BRIEF REVIEW |
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| Year : 2016 | Volume
: 5
| Issue : 3 | Page : 99-103 |
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Role of cyclic AMP and cyclic GMP as modulators of platelet cytosolic calcium
Gundu H.R. Rao PhD
Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, USA
| Date of Web Publication | 26-Sep-2016 |
Correspondence Address:
Gundu H.R. Rao
Laboratory Medicine and Pathology, University of Minnesota, Minneapolis
USA
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/2250-3528.191101
Studies from our laboratory at the University of Minnesota as well as that of others have demonstrated that agonist-mediated receptor stimulation leads to the activation of phospholipase C and formation of second messengers, diacyl glycerol and inositol trisphosphate, and mobilization of cytosolic free calcium. Elevation in intracellular calcium activates phospholipase A2 and release of arachidonic acid. This fatty acid is further converted to active metabolites, prostaglandin endoperoxides, and thromboxanes (TXA 2 ). TXA 2 promote fibrinogen binding, irreversible aggregation, and the release of granule contents, including serotonin and ADP. Agents that promote irreversible aggregation, facilitate fibrinogen binding, and those drugs that dissociate this process do so by lowering the cytosolic calcium levels. Endogenous antagonists such as PGE 1 , PGI 2 , and NO exert their inhibitory effect on platelet function by the action of cyclic nucleotides, cyclic AMP and cyclic GMP. Epinephrine-induced modulation of alpha-adrenergic receptor restores the action of agonists in drug-induced refractory platelets. As far as the role of cyclic nucleotides is considered, the observations are consistent with the concept that cyclic nucleotides participation in the biologic regulation may be modulated by cytoplasmic cations, and that a change in intracellular cyclic nucleotides and appropriate cations serves to promote the participation of both regulatory effectors. Anti-platelet drugs such as aspirin, indomethacin, and PGE 1 do not inhibit platelet interactions with the vessel wall. Based on this collective knowledge, novel antiplatelet drugs should be developed for the prevention of acute vascular events. Keywords: Activation, anti-platelet drugs, cAMP, cGMP calcium, platelet
How to cite this article:
Rao GH. Role of cyclic AMP and cyclic GMP as modulators of platelet cytosolic calcium. J Clin Prev Cardiol 2016;5:99-103 |
| Introduction | |  |
Blood platelets play a very important role in the pathogenesis of atherosclerosis, interaction with the vessel wall, thrombus formation, thrombus growth, and coagulation. In other words, development of thrombotic state involves four main factors; alterations in the vessel wall physiology, the interaction of formed elements of blood, activation of coagulation factors, and blood flow dynamics. In the arterial thrombosis, the major part in the initiation and growth of thrombi is played by platelets and fibrinogen. Given these observations, for the development of drugs that inhibit platelet function, it is helpful to understand the various biochemical mechanisms, which contribute to thrombus formation so that novel anti-platelet drugs could be developed. In this mini-review, we will discuss some of the early discoveries in our laboratory at the University of Minnesota that led us to understand the mechanisms involved in adhesion, shape change, spreading, aggregation, secretion of granule contents, thrombus formation, and thrombus growth. As this is an article that expresses my point-of-view on the topic, I have taken liberties to summarize our work of over three decades. The results of these studies have been presented in various national and international conferences over the years, as such may not be easily accessible. Moreover, it is hard to understand the overall findings without reading all those published articles. Without that collective knowledge, it would be difficult to design novel specific and effective antiplatelet therapies, which would be the subject of my next overview to be published in this journal.
In the early 70s, it was believed that agonists activate platelets by an as yet unknown mechanism, cause release of granule contents, including serotonin, adenosine diphosphate, and calcium. It was assumed that released components acted as recruiting agents as well as potentiators of platelet activation and aggregation. Studies from our laboratory for the first time demonstrated that calcium ionophore (A23187) by moving calcium across the membranes was able to induce aggregation, contraction, as well as release of the internal contents. I would say that it was a remarkable observation. We were able to show in this study that the antibiotic ionophore was able to move calcium across the membrane, facilitate the release of internal membrane-bound calcium, promote the conversion of soluble actin to filamentous actin, and induce contraction and release of the granule contents. We also demonstrated that the agents that elevate cyclic AMP (cAMP) inhibited this process. [1] Further studies from our laboratory demonstrated the similarity between the ionophore-induced platelet activation and that of changes induced by natural platelet agonists such as collagen and thrombin. [2] Now that we knew that elevation of cytosolic calcium was the key to contraction and secretion pathways, we had to explore how agonists mobilize intracellular calcium.
Since we were located close to Hormel, Minnesota, where we had easy access to freshly harvested sheep vesicular glands (SVGs), we were able to obtain these glands and show that one could obtain labile lipid platelet agonists capable of aggregating aspirin-treated platelets. [3] Studies from our laboratory for the first time demonstrated the localization of the prostaglandin synthetase in the platelet-dense tubular system. [4] My associate Jonathan Gerrard using arachidonic acid (AA), endoperoxides, and ionophore as agonists, demonstrated calcium mobilization and phosphorylation of platelet proteins. [5] We were also able to demonstrate agonist-mediated calcium mobilization using calcium-specific flourophores. [6],[7],[8],[9],[10]
During the same period, Prof. Nelson D. Goldberg of the University of Minnesota, published an article, to explain a phenomenon of "Yin/Yang hypothesis," to describe a concept of biological regulation, in which two cellular components, cyclic AMP and cyclic GMP, are envisioned to provide the facilitatory or supportive role in modulating a variety of biological events. [8] In Chinese philosophy, Yin and Yang describes how opposite or contrary forces are actually complementary, interconnected, and interdependent to each other, as they interrelate to one another. We were quite impressed by the work of Prof. Goldberg and were very much interested in demonstrating such an effect in platelet activation and deactivation. Therefore, we collaborated with his group and published an abstract in the Journal of Clinical Investigation in 1973 (I was not able to get this abstract by internet search). This is the way I have summarized our observations in an abstract on my Researchgate site: "In those days when cyclic GMP (cGMP) ruled most cellular events or that is what the experts in Nelson Goldberg's laboratory believed, these studies were done to demonstrate that there was a role for cGMP in modulation of platelet physiology and function. However, later studies demonstrated that elevation of cGMP by "nitroso" compounds, as well as nitric oxide inhibited platelet activation." One exception for this observation is cGMP elevation by ascorbic acid. The ascorbic acid-induced increase in cyclic GMP does not lead to the inhibition of aggregation, the exact role for this observation begs explanation.
Based on our studies, platelet activation story goes something like this. [9],[10],[11],[12],[13],[14],[15],[16],[17],[18],[19],[20],[21],[22],[23],[24],[25],[26],[27],[28],[29],[30] One or more agonists interact with the platelet lipid membrane-associated, glycoprotein-rich receptors [Figure 1]. The agonist interaction with specific receptors results in transmembrane signaling via transmembrane proteins, such as GTP-binding proteins. Receptor-mediated signaling results in the hydrolysis of GTP to GDP and activation of phospholipase C (PLC). This enzyme acts on membrane-associated phospholipids (inositol phosphates) and generates second messengers such as inositol 1, 4'- 5' trisphosphate (IP3) and diacyl-glycerol (2-DG). Inositol trisphosphate mobilizes cytosolic calcium from membrane stores and elevates free cytosolic calcium. This elevation in cytosolic-free calcium activates phospholipase A 2 (PlA 2 ) and releases AA. This fatty acid is further converted by cyclooxygenase (COX) enzymes to prostaglandin endoperoxides (PGH 2 , PGG 2 ) and thromboxanes (TXA 2 ), pro-aggregatory, and vasoconstrictory metabolites. These active metabolites of AA are released to the exterior of the cells and they, in turn, activate specific receptors on platelet membranes. This is one of the common pathways to activate GP11b/111a receptors on platelets. Once these receptors are activated, they can recognize the active binding site on fibrinogen that contains the peptide; Arg-Gly-Asp (R-G-D) sequence of amino acids and brings platelets together (aggregation or thrombus formation). As far as the morphology and cytoskeletal changes are concerned, platelets when activated, change shape, extend pseudopods, spread on the surface, assemble filamentous actin, contract, and secrete granule contents. Series of studies from our laboratory have demonstrated that platelet deactivation is achieved by lowering the elevated levels of cytosolic-free calcium by the second messengers, cAMP and cGMP. [10]  | Figure 1: Steps involved in agonist mediated calcium mobilization in platelets
Click here to view |
By this time, studies from our laboratory as well as that of others had demonstrated that platelet aggregation is dependent on GP11b/111a activation and fibrinogen binding. In a series of elegant studies, we were able to demonstrate that one can dissociate bound fibrinogen from aggregated platelets by lowering cytosolic calcium. [5],[13] These studies further demonstrated that fibrinogen binding which parallels irreversible aggregation is accompanied by phosphorylation of specific proteins, and the disaggregation and reaggregation cycles are accompanied by disassociation and reassociation of fibrinogen and protein phosphorylation cycles [Figure 2]. [5],[14],[15] | Figure 2: Sequential platelet aggregation, disaggregation, and re-aggregation
Click here to view |
Having explained basic mechanisms involved in receptor-mediated activation of PLC, calcium mobilization, stimulation of PLA 2 , release of AA, generation of second messengers, PGG 2 , PGH 2 , TXA 2 , cAMP, cGMP, and modulation of cytosolic calcium, we turned our attention to define the mechanisms involved in adhesion, spreading irreversible aggregation, and platelet-vessel wall interactions. [14],[29] Using platelets of patients with Hermansky-Pudlak syndrome, which are devoid of dense granules, we demonstrated irreversible aggregation independent of release of granule contents. [11],[16] Using COX-deficient platelets, we demonstrated irreversible aggregation independent of prostaglandins. [11],[18],[19] Deploying Quin-2 AM, a calcium flourophore and a chelating agent, we demonstrated adhesion and irreversible aggregation of platelets, independent of cytosolic calcium. [9] Using denuded rabbit aorta, we demonstrated that commonly used antiplatelet drugs do not prevent platelet-vessel wall interactions. [17],[27]
Summarizing the work of four decades in our laboratory at the University of Minnesota in a short overview is a herculean task. However, it is important to understand the salient findings so that we can figure out why some of the anti-platelet drugs failed and some were successful. For instance, collective knowledge from our group as well as that of others regarding the activation of GP11b/111a receptor as a prerequisite for fibrinogen binding, and the sequence of amino acids that are recognized by the activated receptor (R-G-D) played an important role in the development of RGD mimics, as well as the monoclonal antibody Abciximab (ReoPro). [30],[31],[32] Platelets do not recognize this RGD sequence in soluble fibrinogen circulating in the blood; once the GP11b/111a receptor is activated, then they can find this binding site. Whereas once the fibrinogen is bound on a surface, this binding site is exposed and activation of the receptor is not essential for platelets to interact with the surface-bound fibrinogen. Contrary to this phenomenon, platelet GP1b receptor does not need any activation to bind von Willebrand factor protein. However, this globular protein can interact with platelets only at very high shear force (arterial circulation). Our studies very clearly demonstrated that drug-induced refractoriness of platelets can be overcome by the action of multiple agonists or by epinephrine-induced mechanism of membrane modulation. [19],[20],[21],[22],[23],[24],[25],[26],[31],[32],[33],[34],[35] Since platelet interactions with the vessel wall was mediated by extracellular matrix components, commonly used anti-platelet drugs such as aspirin, indomethacin, and PGE 1 failed to inhibit these interactions. [12],[25],[27]
From the very first report from our laboratory in the 70s, in which we had demonstrated a raise in cGMP in parallel with irreversible aggregation and release of granule contents, there has been considerable conflicting reports on the role of cyclic nucleotides in platelet function. [8],[36],[37] Noble Laureate Louis Ignarro in his chapter on the regulation of polymorphonuclear leukocyte, macrophage, and platelet function writes, "recent advances in the fields of cyclic nucleotide and biological regulation have made it clear, however, that a second cyclic nucleotide cyclic GMP, serves as important, and perhaps obligatory function in the bio regulation of cellular processes (Refers to our 1973 abstract). Much of the work involving cGMP suggests that it may function to mediate or signal cellular processes that are ultimately antagonistic or opposite in direction to those mediated by cAMP. [38]" Studies from our laboratory as well as that of others have demonstrated the inhibitory role of agents that elevate cAMP as well as cGMP. [20],[21],[22],[37] According to the recent studies, the activation of human platelets seems to be inhibited by two intracellular pathways, regulated by either cAMP or cGMP-elevating agents. [39] These studies have demonstrated that inhibitory effect of these antagonists has to be mediated by the phosphorylation of vasodilator-stimulated phosphoprotein (VASP). Our results on the inhibitory role of cGMP on platelet function, similar to that of cAMP, has been confirmed by studies from many other laboratories including the Welcome Laboratories under the leadership of Salvador Moncada. [40],[41]
| Conclusion | | |
Studies from our laboratory at the University of Minnesota as well as that of others have demonstrated that agonist-mediated receptor stimulation leads to the activation of PLC and formation of second messengers, diacyl glycerol and inositol trisphosphate, and mobilization of cytosolic-free calcium. Elevation in intracellular calcium activates PlA 2 and release of AA. This fatty acid is further converted into active metabolites, PGH 2 , PGG 2 , and TXA 2 . TXA 2 promote fibrinogen binding, irreversible aggregation, and the release of granule contents, including serotonin and ADP. Agents that promote irreversible aggregation, facilitate fibrinogen binding, and those drugs that dissociate this process do so by lowering the cytosolic calcium levels. Endogenous antagonists such as PGE 1 , PGI 2 , and NO exert their inhibitory effect on platelet function by the action of cyclic nucleotides, cAMP and cGMP. Epinephrine-induced modulation of alpha-adrenergic receptor restores the action of agonists in drug-induced refractory platelets. As far as the role of cyclic nucleotides is considered, the observations are consistent with the concept that cyclic nucleotides participation in the biologic regulation may be modulated by cytoplasmic cations, and that a change in intracellular cyclic nucleotides and appropriate cations serves to promote the participation of both regulatory effectors. [42] Anti-platelet drugs such as aspirin, indomethacin, and PGE 1 do not inhibit platelet interactions with the vessel wall. Based on this collective knowledge, novel antiplatelet drugs should be developed for the prevention of acute vascular events.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
| References | | |
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[Figure 1], [Figure 2]
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