Summary
Background
Genetic variations that affect the structure of the thromboxane A2 receptor (TP receptor) provide insights into the function of this key platelet and vascular receptor, but are ...very rare in unselected populations.
Objectives
To determine the functional consequences of the TP receptor Trp29Cys (W29C) substitution.
Patients/Methods
We performed a detailed phenotypic analysis of an index case (P1) with reduced platelet aggregation and secretion responses to TP receptor pathway activators, and a heterozygous TP receptor W29C substitution. An analysis of the variant W29C TP receptor expressed in heterologous cells was performed.
Results
Total TP receptor expression in platelets from P1 was similar to that of controls, but there was reduced maximum binding and reduced affinity of binding to the TP receptor antagonist 3HSQ29548. HEK293 cells transfected with W29C TP receptor cDNA showed similar total TP receptor expression to wild‐type (WT) controls. However, the TP receptor agonist U46619 was less potent at inducing rises in cytosolic free Ca2+ in HEK293 cells expressing the W29C TP receptor than in WT controls, indicating reduced receptor function. Immunofluorescence microscopy and cell surface ELISA showed intracellular retention and reduced cell surface expression of the W29C TP receptor in HEK293 cells. Consistent with the platelet phenotype, both maximum binding and the affinity of binding of 3HSQ29548 to the W29C TP receptor were reduced compared to WT controls.
Conclusion
These findings extend the phenotypic description of the very rare disorder TP receptor deficiency, and show that the W29C substitution reduces TP receptor function by reducing surface receptor expression and by disrupting ligand binding.
Data analysis workflows in many scientific domains have become increasingly complex and flexible. Here we assess the effect of this flexibility on the results of functional magnetic resonance ...imaging by asking 70 independent teams to analyse the same dataset, testing the same 9 ex-ante hypotheses
. The flexibility of analytical approaches is exemplified by the fact that no two teams chose identical workflows to analyse the data. This flexibility resulted in sizeable variation in the results of hypothesis tests, even for teams whose statistical maps were highly correlated at intermediate stages of the analysis pipeline. Variation in reported results was related to several aspects of analysis methodology. Notably, a meta-analytical approach that aggregated information across teams yielded a significant consensus in activated regions. Furthermore, prediction markets of researchers in the field revealed an overestimation of the likelihood of significant findings, even by researchers with direct knowledge of the dataset
. Our findings show that analytical flexibility can have substantial effects on scientific conclusions, and identify factors that may be related to variability in the analysis of functional magnetic resonance imaging. The results emphasize the importance of validating and sharing complex analysis workflows, and demonstrate the need for performing and reporting multiple analyses of the same data. Potential approaches that could be used to mitigate issues related to analytical variability are discussed.
Platelet‐expressed GPCRs are critical regulators of platelet function. Pharmacological blockade of these receptors forms a powerful therapeutic tool in the treatment and prevention of arterial ...thrombosis associated with coronary atherosclerosis and ischaemic stroke. However, anti‐thrombotic drug therapy is associated with high inter‐patient variability in therapeutic response and adverse bleeding side effects. In order to optimize the use of existing anti‐platelet drugs and to develop new therapies, more detailed knowledge is required relating to the molecular mechanisms that regulate GPCR and therefore platelet function. One approach has been to identify rare, function‐disrupting mutations within key platelet proteins in patients with bleeding disorders. In this review, we describe how an integrated functional genomics strategy has contributed important structure‐function information about platelet GPCRs with specific emphasis upon purinergic and thromboxane A2 receptors. We also discuss the potential implications these findings have for pharmacotherapy and for understanding the molecular basis of mild bleeding disorders.
Linked Articles
This article is part of a themed section on 5th BPS Focused Meeting on Cell Signalling. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue‐13
Interleukin-1 beta (IL-1 beta)-converting enzyme cleaves the IL-1 beta precursor to mature IL-1 beta, an important mediator of inflammation. The identification of the enzyme as a unique cysteine ...protease and the design of potent peptide aldehyde inhibitors are described. Purification and cloning of the complementary DNA indicates that IL-1 beta-converting enzyme is composed of two nonidentical subunits that are derived from a single proenzyme, possibly by autoproteolysis. Selective inhibition of the enzyme in human blood monocytes blocks production of mature IL-1 beta, indicating that it is a potential therapeutic target.
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