Platelets are indispensable for primary haemostasis, but their function needs to be tightly regulated to prevent excessive platelet activity, possibly leading to atherothrombotic events. An important mediator of the platelet activity is cyclic AMP (cAMP), which inhibits platelet aggregation. Intracellular cAMP levels are regulated via the Gs and Gi alpha subunits of heterotrimeric G proteins, which couple to adenylyl cyclase to respectively stimulate or inhibit cAMP production. Binding of a ligand to its G protein-coupled seven-transmembrane receptor activates these G proteins. In this review, we discuss a Gs-coupled receptor on platelets, VPAC1, and 2 important Gi-coupled receptors, the ADP receptor P2Y(12) and the prostaglandin E(2) receptor EP3. The regulation of platelet cAMP levels at the level of the receptors themselves or the G proteins coupled to them is analyzed. Alterations in Gsα and Giα function are associated with altered platelet reactivity. An increase in Gs function, or alternatively a defective Gi signaling, can be a risk factor for bleeding, while a loss of Gs function can result in a prothrombotic state. Regulator of G protein signaling (RGS) proteins accelerate the rate of inactivation of G protein-mediated signaling. One of the RGS proteins, RGS2, inhibits Gs signaling by interacting directly with adenylyl cyclase. The thienopyridine class of antiplatelet agents is based on cAMP-mediated regulation of platelet function through modification of the P2Y(12) receptor. Clopidogrel and some other novel cAMP regulators are discussed. Secondly, we review the use of prostacyclin derivatives to treat pulmonary arterial hypertension.
Platelet Gs hypofunction and abnormal morphology resulting from a heterozygous RGS2 mutation. J Thromb Haemost 2010; 8: 1594-603.Summary. Background: Regulator of G-protein signaling (RGS) 2 negatively regulates Gs signaling by inhibiting the activation of adenylyl cyclase (AC). RGS2 mRNA contains four translation initiation sites, leading to four isoforms with different abilities to inhibit AC activity; the largest isoform is the most pronounced inhibitor. A role for RGS2 in platelets is not known. Objective: To describe a heterozygous RGS2 mutation (G23D) in three related patients, leading to Gs hypofunction in their platelets, and to study the mechanism behind the effect of the RGS2 mutation on platelet function and morphology. Methods: Gs signaling was studied ex vivo in platelets and in vitro in transfected cells. Translation initiation was evaluated in vitro, and the interaction of wild-type and G23D RGS2 with AC was unraveled via immunoprecipitation. Platelet granule content was analyzed with proteomics. Results: The mutation leads to reduced cAMP production after stimulation of Gscoupled receptors. The largest RGS2 isoforms, with strong AC inhibitor activity, are enriched when the mutation is present, as compared with wild-type RGS2. Moreover, the mutation results in a stronger interaction of RGS2 with AC. G23D RGS2 carriers have enlarged, round platelets with abnormal a-granules. Proteomics of the platelet releasate revealed altered expression of some proteins involved in actin assembly, and carriers seemed to have a reduced platelet shape change. Conclusions: We present the first platelet Gs signaling defect caused by a heterozygous RGS2 variant that results in a unique mutational mechanism, such as the differential use of translation initiation sites resulting in different functional RGS2 isoforms.
Regulator of G protein signaling (RGS) proteins stimulate the GTPase activity of Gα subunits of heterotrimeric G proteins, thereby negatively regulating G protein signaling. In this way, RGS2 acts as a negative regulator of Gq and Gi signaling. It has also been described as a negative regulator of Gs signaling, but via a different mechanism. It inhibits the activation of adenylyl cyclase (AC), the target molecule of Gs, by interacting with it. In olfactory neurons, it was shown that RGS2 attenuates activation of AC type III (Sinnarajah et al., Nature 2001), the main AC subtype in platelets. In this study, we describe the first human genetic defect in RGS2 and provide evidence that RGS2 influences the cAMP level in platelets after Gs stimulation. The proposita is an obese 16-year-old girl with borderline IQ, hirsutism and an increased bone alkaline phosphatase. These symptoms are similar to features of Albright hereditary osteodystrophy, due to heterozygous inactivating mutations in the Gsα gene. The Gsα gene of the proposita is normal, but she carries a missense mutation in the RGS2 gene, resulting in a Gly to Asp substitution in the conserved residue 23 (G23D). This substitution could also be found in her mother and brother, but not in 200 unrelated normal controls. The family members carrying this mutation present with a relatively low number of platelets (+/−150.000/μL) and an increased mean platelet volume (+/−13 fL) and platelet distribution width (+/−17.5 %). Also, platelet function is affected by the mutation. Platelet aggregation is normal in response to all standard agonists, but when the Gs pathway is challenged in their platelets, high levels of different Gs agonists are needed to get inhibition of aggregation in comparison to controls or the father. We also measured cAMP levels in platelets and found that stimulation of the Gs coupled receptors with Gs agonists produced less cAMP in the affected family members. The functional relevance of the mutation was further studied in vitro in HEK293 and MEG-01 cells transfected with wildtype RGS2 and RGS2-G23D. cAMP levels were measured at different time points after stimulation of these cells with Gs agonists. These measurements show that cAMP levels are lower in cells transfected with RGS2-G23D, compared to wildtype RGS2. This indicates that the reduction in cAMP levels found in the platelets of the affected members, is a functional consequence of the mutation. To understand why this mutation leads to an altered function of RGS2, we studied the effect of the mutation at the protein level. Recently, it was shown that there are 4 different translation initiation sites in the RGS2 mRNA, giving rise to 4 proteins with different functional characteristics (Gu et al., Mol Pharmacol 2008). An in vitro transcriptiontranslation assay showed that the presence of the mutation results in a different protein expression profile. We excluded a difference in posttranslational modifications to be the cause of this divergent pattern. The G23D mutation is located in the proximity of 2 of the different translation initiation sites and its presence alters the use of these sites. This results in a different expression profile of the functionally different RGS2 proteins. In conclusion, we present the first platelet Gs signaling defect due to an RGS2 mutation associated with aberrant RGS2 translation.
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