Key Points• Ticagrelor acts as an inverse agonist at the P2Y 12 R, inhibiting basal agonistindependent signaling. • Ticagrelor inhibits the adenosine transporter ENT1 not only on erythrocytes, but on platelets too.Ticagrelor is a potent antagonist of the P2Y 12 receptor (P2Y 12 R) and consequently an inhibitor of platelet activity effective in the treatment of atherothrombosis. Here, we sought to further characterize its molecular mechanism of action. Initial studies showed that ticagrelor promoted a greater inhibition of adenosine 59-diphosphate (ADP)-induced Ca 21 release in washed platelets vs other P2Y 12 R antagonists. This additional effect of ticagrelor beyond P2Y 12 R antagonism was in part as a consequence of ticagrelor inhibiting the equilibrative nucleoside transporter 1 (ENT1) on platelets, leading to accumulation of extracellular adenosine and activation of G s -coupled adenosine A 2A receptors. This contributed to an increase in basal cyclic adenosine monophosphate (cAMP) and vasodilator-stimulated phosphoprotein phosphorylation (VASP-P). In addition, ticagrelor increased platelet cAMP and VASP-P in the absence of ADP in an adenosine receptor-independent manner. We hypothesized that this increase originated from a direct effect on basal agonist-independent P2Y 12 R signaling, and this was validated in 1321N1 cells stably transfected with human P2Y 12 R. In these cells, ticagrelor blocked the constitutive agonist-independent activity of the P2Y 12 R, limiting basal G i -coupled signaling and thereby increasing cAMP levels. These data suggest that ticagrelor has the pharmacological profile of an inverse agonist. Based on our results showing insurmountable inhibition of ADP-induced Ca 21 release and forskolin-induced cAMP, the mode of antagonism of ticagrelor also appears noncompetitive, at least functionally. In summary, our studies describe 2 novel modes of action of ticagrelor, inhibition of platelet ENT1 and inverse agonism at the P2Y 12 R that contribute to its effective inhibition of platelet activation. (Blood. 2016;128(23):2717-2728

Morel-Kopp M-C. Receptor homodimerization plays a critical role in a novel dominant negative P2RY12 variant identified in a family with severe bleeding. J Thromb Haemost 2018; 16: 44-53. Essentials• Three dominant variants for the autosomal recessive bleeding disorder type-8 have been described. • To date, there has been no phenotype/genotype correlation explaining their dominant transmission.• Proline plays an important role in P2Y12R ligand binding and signaling defects.• P2Y12R homodimer formation is critical for the receptor function and signaling.Summary. Background: Although inherited platelet disorders are still underdiagnosed worldwide, advances in molecular techniques are improving disease diagnosis and patient management. Objective: To identify and characterize the mechanism underlying the bleeding phenotype in a Caucasian family with an autosomal dominant P2RY12 variant. Methods: Full blood counts, platelet aggregometry, flow cytometry and western blotting were performed before next-generation sequencing (NGS). Detailed molecular analysis of the identified variant of the P2Y12 receptor (P2Y12R) was subsequently performed in mammalian cells overexpressing receptor constructs. Results: All three referred individuals had markedly impaired ADP-induced platelet aggregation with primary wave only, despite normal total and surface P2Y12R expression. By NGS, a single P2RY12:c.G794C substitution (p.R265P) was identified in all affected individuals, and this was confirmed by Sanger sequencing. Mammalian cell experiments with the R265P-P2Y12R variant showed normal receptor surface expression versus wild-type (WT) P2Y12R. Agonist-stimulated R265P-P2Y12R function (both signaling and surface receptor loss) was reduced versus WT P2Y12R. Critically, R265P-P2Y12R acted in a dominant negative manner, with agonist-stimulated WT P2Y12R activity being reduced by variant coexpression, suggesting dramatic loss of WT homodimers. Importantly, platelet P2RY12 cDNA cloning and sequencing in two affected individuals also revealed three-fold mutant mRNA overexpression, decreasing even further the likelihood of WT homodimer formation. R265 located within extracellular loop 3 (EL3) is one of four residues that are important for receptor functional integrity, maintaining the binding pocket conformation and allowing rotation following ligand binding. Conclusion: This novel dominant negative variant confirms the important role of R265 in EL3 in the functional integrity of P2Y12R, and suggests that pathologic heterodimer formation may underlie this family bleeding phenotype.

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BACKGROUND Viscoelastic tests (VETs) are used widely to monitor hemostasis in settings such as cardiac surgery. There has also been renewed interest in cold stored platelets (CSPs) to manage bleeding in this setting. CSPs are reported to have altered hemostatic properties compared to room temperature platelets (RTPs), including activation of GPIIb/IIIa. We investigated whether the functional differences between CSP and RTP affected the performance of the PlateletMapping VET on the TEG 5000 and 6s analyzer. METHOD Platelet concentrates were divided equally into CSP (stored at 4°C ± 2°C) and RTP (stored at 22°C ± 2°C) fractions. Whole blood was treated to induce platelet dysfunction (WBIPD) by incubating with anti‐platelet drugs (1.0 μM ticagrelor and 10 μM aspirin) or by simulating cardiopulmonary bypass. WBIPD samples were then mixed with 20% by volume of CSPs or RTPs to model platelet transfusion before analysis using the PlateletMapping VET. RESULTS Addition of CSPs to WBIPD increased the PlateletMapping MAFIBRIN and MAADP parameters with the TEG 5000 analyzer (both p < 0.0001 compared to addition of buffer alone). This effect was not observed with RTPs. The differential effect of CSPs on the MAFIBRIN corrected after pre‐incubation with the GPIIb/IIIa antagonist tirofiban and was quantitatively less with the PlateletMapping test for the TEG 6s analyzer which contains the GPIIb/IIa antagonist abciximab. DISCUSSION The PlateletMapping MAFIBRIN and MAADP test results may be misleadingly high with CSPs, particularly with the TEG 5000 analyzer, most likely due to constitutive activation of GPIIb/IIIa on CSPs during storage. TEG PlateletMapping results should be interpreted with caution following CSP transfusion.

The reactivity of platelets, which play a key role in the pathogenesis of atherothrombosis, is tightly regulated. The integral membrane protein tetherin/bone marrow stromal antigen-2 (BST-2) regulates membrane organization, altering both lipid and protein distribution within the plasma membrane. Because membrane microdomains have an established role in platelet receptor biology, we sought to characterize the physiological relevance of tetherin/BST-2 in those cells. To characterize the potential importance of tetherin/BST-2 to platelet function, we used tetherin/BST-2−/− murine platelets. In the mice, we found enhanced function and signaling downstream of a subset of membrane microdomain–expressing receptors, including the P2Y12, TP thromboxane, thrombin, and GPVI receptors. Preliminary studies in humans have revealed that treatment with interferon-α (IFN-α), which upregulates platelet tetherin/BST-2 expression, also reduces adenosine diphosphate–stimulated platelet receptor function and reactivity. A more comprehensive understanding of how tetherin/BST-2 negatively regulates receptor function was provided in cell line experiments, where we focused on the therapeutically relevant P2Y12 receptor (P2Y12R). Tetherin/BST-2 expression reduced both P2Y12R activation and trafficking, which was accompanied by reduced receptor lateral mobility specifically within membrane microdomains. In fluorescence lifetime imaging-Förster resonance energy transfer (FLIM-FRET)–based experiments, agonist stimulation reduced basal association between P2Y12R and tetherin/BST-2. Notably, the glycosylphosphatidylinositol (GPI) anchor of tetherin/BST-2 was required for both receptor interaction and observed functional effects. In summary, we established, for the first time, a fundamental role of the ubiquitously expressed protein tetherin/BST-2 in negatively regulating membrane microdomain–expressed platelet receptor function.

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We describe the clinical, muscle and nerve biopsy, and genetic findings in a 10-year-old girl with a profound and rapid global regression. She presented during neonatal period with hypotonia, followed by weakness in the facial, bulbar, respiratory, and neck flexor muscles. She developed bilateral cataracts at 4 months of age and started to regress. Quadriceps muscle biopsy revealed extensive fiber atrophy but sparing of some, predominantly type 1, fibers. Sural nerve biopsy showed depletion of myelinated and unmyelinated fibers; most remaining myelinated fibers were of small caliber. Neuroimaging revealed global brain atrophy. Although the investigations indicated a multisystem disorder, extensive genetic and metabolic investigations were negative. She was tracheostomy- and ventilator-dependent for most of her life. The child died at 10 years of age. Further deoxyribonucleic acid analysis undertaken via whole genome sequencing revealed a novel pathogenic GFER sequence variant consistent with the patient's clinical presentation.