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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., (© 2021 by The American Society of Hematology.)

Background To assess differences in platelet inhibition during ticagrelor monotherapy (TIC) or dual therapy with ticagrelor and aspirin (TIC+ASP) in patients after percutaneous coronary intervention using a comprehensive panel of functional tests. Methods and Results In a single-center parallel group, open label, randomized controlled trial, 110 participants were randomized to receive either TIC (n=55) or TIC+ASP (n=55) for 4 weeks. The primary outcome was the platelet aggregation response with 10 μmol/L thrombin receptor activation peptide-6 (TRAP-6). The secondary outcomes were platelet aggregation responses and binding of surface activation markers with a panel of other activators. The mean percentage aggregation for 10 μmol/L TRAP-6 was similar for the TIC and TIC+ASP groups (mean difference+4.29; 95% CI, -0.87 to +9.46). Aggregation was higher in the TIC group compared with the TIC+ASP group with 1 μg/mL (+6.47; +2.04 to +10.90) and 0.5 μg/mL (+14.00; +7.63 to +20.39) collagen related peptide. Aggregation responses with 5 μmol/L TRAP-6, 5 μmol/L or 2.5 μmol/L thromboxane A 2 receptor agonist and surface activation marker binding with 5 μmol/L TRAP-6 or 0.5 μg/mL collagen related peptide were the same between the treatment groups. Conclusions Patients with PCI show similar levels of inhibition of most platelet activation pathways with TIC compared with dual therapy with TIC + ASP. However, the greater aggregation response with collagen related peptide during TIC indicates incomplete inhibition of glycoprotein VI (collagen) receptor-mediated platelet activation. This difference in pharmacodynamic response to anti-platelet medication may contribute to the lower bleeding rates observed with TIC compared with dual antiplatelet therapy in recent clinical trials. Registration Information URL: https://www.isrctn.com; Unique Identifier ISRCTN84335288.

Carbonic anhydrase (CA) inhibitors have a long history of safe clinical use as mild diuretics, in the treatment of glaucoma and for altitude sickness prevention. In this study, we aimed to determine if CA inhibition may be an alternative approach to control thrombosis. We utilized a high-resolution dynamic imaging approach to provide mechanistic evidence that CA inhibitors may be potent anti-procoagulant agents in vitro and effective anti-thrombotics in vivo . Acetazolamide and methazolamide, while sparing platelet secretion, attenuated intracellular chloride ion entry and suppressed the procoagulant response of activated platelets in vitro and thrombosis in vivo . The chemically similar N-methyl acetazolamide, which lacks CA inhibitory activity, did not affect platelet procoagulant response in vitro . Outputs from rotational thromboelastometry did not reflect changes in procoagulant activity and reveal the need for a suitable clinical test for procoagulant activity. Drugs specifically targeting procoagulant remodeling of activated platelets, by blockade of carbonic anhydrases, may provide a new way to control platelet-driven thrombosis without blocking essential platelet secretion responses.

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 MA FIBRIN and MA ADP 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 MA FIBRIN 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 MA FIBRIN and MA ADP 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., (© 2020 Crown copyright.Transfusion © 2020 AABB.)

Introduction: Protein C (PC) deficiency results in dysregulated thrombin generation and increases thrombosis risk., Methods: In order to investigate the potential effects of anticoagulant drugs in PC deficiency, we evaluated the pharmacodynamic effect of selective direct factor (F) IIa inhibitors (dabigatran and argatroban), selective direct FXa inhibitors (rivaroxaban and apixaban) and an indirect FXa/FIIa inhibitor (enoxaparin) in commercial PC-deficient plasma using thrombin generation and viscoelastometry assays modified to reflect PC anticoagulant activity., Results: Endogenous thrombin potential (ETP) and peak thrombin concentration (PTC) were increased in PC-deficient plasma but this corrected completely with PC concentrate. Inhibition of FIIa and FXa with the selective inhibitors also corrected the increased ETP and PTC but required high drug concentrations. There was sustained low-level thrombin generation in PC-deficient plasma with FXa inhibitors but not with FIIa inhibitors. Adding PC concentrate to PC-deficient plasma anticoagulated with dabigatran had little additional effect on ETP or PTC. In contrast, addition of even small quantities of PC concentrate to PC-deficient plasma anticoagulated with rivaroxaban further diminished ETP, primarily by abolishing sustained thrombin generation. In the viscoelastometry assay, the coagulation time was shortened and α-angle increased in PC-deficient plasma. These abnormalities reversed with both dabigatran and rivaroxaban., Conclusion: The selective direct FXa and FIIa inhibitors at high concentrations both counteracted the abnormal thrombin generation and clot formation observed in PC-deficient plasma, but with qualitative differences in their effects., (© 2019 John Wiley & Sons Ltd.)

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., (© 2017 International Society on Thrombosis and Haemostasis.)

Genetic variations in G protein-coupled receptor (GPCR) genes can disrupt receptor function in a wide variety of human genetic diseases, including platelet bleeding disorders. Platelets are critical for haemostasis with inappropriate platelet activation leading to the development of arterial thrombosis, which can result in heart attack and stroke whilst decreased platelet activity is associated with an increased risk of bleeding. GPCRs expressed on the surface of platelets play key roles in regulating platelet activity and therefore function. Receptors include purinergic receptors (P2Y 1 and P2Y 12 ), proteinase-activated receptor (PAR1 and PAR4) and thromboxane receptors (TPα), among others. Pharmacological blockade of these receptors forms a powerful therapeutic tool in the treatment and prevention of arterial thrombosis. With the advance of genomic technologies, there has been a substantial increase in the identification of naturally occurring rare and common GPCR variants. These variants include single-nucleotide polymorphisms (SNPs) and insertion or deletions that have the potential to alter GPCR expression or function. A number of defects in platelet GPCRs that disrupt receptor function have now been characterized in patients with mild bleeding disorders. This review will focus on rare, function-disrupting variants of platelet GPCRs with particular emphasis upon mutations in the P2Y 12 receptor gene that affect receptor traffic to modulate platelet function. Further this review will outline how the identification and characterization of function-disrupting GPCR mutations provides an essential link in translating our detailed understanding of receptor traffic and function in cell line studies into relevant human biological systems., (Copyright © 2017. Published by Elsevier B.V.)

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 5'-diphosphate (ADP)-induced Ca 2+ 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 2+ 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., (© 2016 by The American Society of Hematology.)