Cyclic nucleotides are important inhibitory regulators of platelet function. These second messengers are hydrolysed by cyclic 3' ,5'-nucleotide phosphodiesterases (PDEs). Three PDEs have been detected in human platelets: cGMP-stimulated phosphodiesterase (PDE2), cGMP-inhibited phosphodiesterase (PDE3), and cGMP-binding, cGMP-selective phosphodiesterase (PDE5). This research investigates the contribution of PDE2 to the regulation of platelet cyclic nucleotide concentrations, and the effects that PDE2 activity has on the inhibition by cAMP and cGMP of platelet aggregation in response to thrombin or collagen. Increases in platelet cAMP were initiated by stimulation of adenylyl cyclase with prostacyclin (PGI₂), whereas the accumulation of cGMP was induced by nitroprusside (NP). The contributions of PDE2 to the hydrolysis of these cyclic nucleotides were evaluated using a novel inhibitor of the enzyme, 𝘦𝘳𝘺𝘵𝘩𝘳𝘰-9-(2-hydroxy-3-nonyl)adenine (EHNA). Before EHNA was used in experiments on platelet function, its effects on partially purified preparations of the three platelet PDEs were studied. These investigations demonstrated that EHNA is a selective and potent inhibitor of platelet PDE2, and indicated that this compound is a more effective inhibitor of cAMP hydrolysis in the presence than in the absence of cGMP. To measure changes in cyclic nucleotide concentrations, platelets were preincubated with [³H]adenine and ³H]guanine to label the metabolic nucleotide pools. NP caused large concentration-dependent increases in platelet [³H] cGMP levels, and this was associated with highly significant but much smaller increases in [³H] cAMP accumulation, which were optimal with 10 μM NP. Higher concentrations of NP had much less effect on platelet [³H] cAMP. A previous study had shown that the increases in platelet cAMP caused by NP were attributable to the inhibition of PDE3 by cGMP (Maurice and Haslam, 1990a), but the inhibitory component observed with high concentrations of NP had not been explained. The present research showed that the accumulation of cAMP and cGMP induced by high NP concentrations is enhanced by EHNA, and so provides the first demonstration that PDE2 activity restricts NP-induced cyclic nucleotide accumulation. To assess whether these changes in platelet cyclic nucleotide levels were important, platelet aggregation in response to thrombin and collagen was monitored. In these studies, EHNA markedly increased the inhibitory action of NP on platelet aggregation. All the effects of NP on cyclic nucleotide accumulation and on platelet aggregation were blocked by a guanylyl cyclase inhibitor, 1𝘏-[1,2,4] oxadiazolo [4,3-α] quinoxalin-1-one, confirming that NP acts solely through activation of this enzyme and that the increases in cAMP are secondary to cGMP formation. However, experiments with the adenylyl cyclase inhibitor, 2',5'-dideoxyadenosine, which diminished the accumulation of cAMP but not that of cGMP, indicated that the inhibition of platelet aggregation is more closely correlated with the increases in cAMP than with those in cGMP. In experiments in which platelet PDE3 was selectively blocked by lixazinone, the accumulation of [³H]cAMP was greatly increased and a corresponding inhibition of thrombin-induced platelet aggregation was observed. Both of these effects were greatly diminished when PDE2 was stimulated by NP (or cGMP). This research demonstrates for the first time that activation of PDE2 by cGMP has marked effects on platelet function, restricting the inhibition of platelet aggregation by agents that increase platelet cAMP. To investigate the importance of PDE2 in regulating different platelet cAMP levels, the effects of EHNA were studied in the presence of 1 or 20 nM PGI₂. Whereas no significant increase in cAMP accumulation was caused by EHNA in the presence of 1 nM PGI₂, at the higher PGI₂ concentration a marked increase was detected when PDE2 was inhibited. NP potentiated the increase in cAMP seen with low PGI₂ but inhibited that seen with a high PGI₂ concentration, indicating a shift in the relative importance of PDE3 and PDE2 as platelet cAMP was increased. These studies show that in the presence of a high concentration of cAMP alone, or of regulatory cGMP, PDE2 makes a major contribution to the hydrolysis of platelet cAMP. Moreover, the results suggest that PDE2 inhibitors could be of value in the therapeutic modification of platelet responses. / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22712 |
| Date | 08 1900 |
| Creators | Dickinson, Natalie |
| Contributors | Haslam, R. J., Biochemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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