Compartmentation and compartment-specific regulation of PDE5 by protein kinase G allows selective cGMP-mediated regulation of platelet functions

Abstract

It is generally accepted that nitric oxide (NO) donors, such as sodium nitroprusside (SNP), or phosphodiesterase 5 (PDE5) inhibitors, including sildenafil, each impact human platelet function. Although a strong correlation exists between the actions of NO donors in platelets and their impact on cGMP, agents such as sildenafil act without increasing global intra-platelet cGMP levels. This study was undertaken to identify how PDE5 inhibitors might act without increasing cGMP. Our data identify PDE5 as an integral component of a protein kinase G1␤ (PKG1␤)-containing signaling complex, reported previously to coordinate cGMP-mediated inhibition of inositol-1, 4, 5-trisphosphate receptor type 1 (IP 3R1)-mediated Ca 2؉ -release. PKG1␤ and PDE5 did not interact in subcellular fractions devoid of IP 3R1 and were not recruited to IP3R1-enriched membranes in response to cGMP-elevating agents. Activation of platelet PKG promoted phosphorylation and activation of the PDE5 fraction tethered to the IP 3R1-PKG complex, an effect not observed for the nontethered PDE5. Based on these findings, we elaborate a model in which PKG selectively activates PDE5 within a defined microdomain in platelets and propose that this mechanism allows spatial and temporal regulation of cGMP signaling in these cells. Recent reports indicate that sildenafil might prove useful in limiting in-stent thrombosis and the thrombotic events associated with the acute coronary syndromes (ACS), situations poorly regulated with currently available therapeutics. We submit that our findings may define a molecular mechanism by which PDE5 inhibition can differentially impact selected cellular functions of platelets, and perhaps of other cell types.calcium ͉ PKG

Apelin: an antithrombotic factor that inhibits platelet function

“…27 Incubation of platelets with the NOS inhibitor N G -monomethyl-L-arginine (1 mM) blocked the rise of cGMP in platelets exposed to apelin ( Figure 3C), suggesting the involvement of NO in cGMP production induced by apelin in platelets. Compared with sodium nitroprusside (10 mM), an NO donor reported to inhibit platelet aggregation, 28 the use of similar concentration of apelin (10 mM) was twofold-less efficient in the induction of cGMP ( Figure 3D). …”

A Phase I proof-of-concept and safety trial of sildenafil to treat cerebral vasospasm following subarachnoid hemorrhage

“…44,60,64,65 The development of platelet-induced microthrombi within the microvasculature has also been implicated in DCI, [20][21][22]63,70 and sildenafil has been shown to limit this prothrombotic cascade. 30,45,62,68,83 Lastly, when cerebral blood flow to an area decreases to ischemic levels, sildenafil has demonstrated the ability to decrease the size of the infarct and improve outcomes by promoting angiogenesis in hypoxic areas. 14,66,[87][88][89] Each of these pathways represents future areas of study in determining sildenafil's role in treating DCI.…”

“…56 Beyond this, there are data suggesting that sildenafil beneficially affects additional contributors of brain injury after SAH. In studies of SAH in animals, sildenafil has shown to reduce plasma levels of endothelin-1, 9,25,26,44,47,55,60,64,65 limit platelet induced microthrombosis, 30,45,62,68,83 and promote cerebral angiogenesis in hypoxic areas. 14,66,[87][88][89] Sildenafil has been extensively studied and proven safe in both healthy and cardiovascularly ill patients.…”

Defining the Phosphodiesterase Superfamily Members in Rat Brain Microvessels

“…24À27 A single cell type can express several different PDEs and the nature and localization of these PDEs is likely to be of major regulatory importance for the local intracellular concentrations of cAMP and/or cGMP. 27 Localized PDEs may modulate the three-dimensional shape, amplitude, and temporal duration of cyclic nucleotides in selective cellular compartments. 27 In total, 246 transcripts displayed differential expression (p < 0.05, 2-folds).…”

“…27 Localized PDEs may modulate the three-dimensional shape, amplitude, and temporal duration of cyclic nucleotides in selective cellular compartments. 27 In total, 246 transcripts displayed differential expression (p < 0.05, 2-folds). Noticeably, a number of these transcripts were relevant to cell growth: Rattus norvegicus platelet-derived growth factor receptor beta (Pdgfrb); platelet-derived growth factor A-chain (PDGF A-chain); transforming growth factor beta stimulated clone 22; Rattus norvegicus VGF nerve growth factor; stromal cell-derived factor 1; transcription elongation factor A2; transcription elongation factor A (SII); and Kruppel-like factor.…”