Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 52
Filter
1.
Br J Pharmacol ; 2024 Jun 05.
Article in English | MEDLINE | ID: mdl-38840293

ABSTRACT

BACKGROUND AND PURPOSE: Thromboxane A2 (TXA2) is a prostanoid produced during platelet activaton, important in enhancing platelet reactivity by activation of TP receptors. However, due to the short half-life, studying TXA2 signalling is challenging. To enhance our understanding of TP receptor-mediated platelet biology, we therefore synthesised mono and difluorinated TXA2 analogues and explored their pharmacology on heterologous and endogenously expressed TP receptor function. EXPERIMENTAL APPROACH: Platelet functional and signalling responses were studied using aggregometry, Ca2+ mobilisation experiments and immunoblotting and compared with an analogue of the TXA2 precursor prostaglandin H2, U46619. Gαq/Gαs receptor signalling was determined using a bioluminescence resonance energy transfer (BRET) assay in a cell line overexpression system. KEY RESULTS: BRET studies revealed that F-TXA2 and F2-TXA2 promoted receptor-stimulated TP receptor G-protein activation similarly to U46619. Unexpectedly, F2-TXA2 caused reversible aggregation in platelets, whereas F-TXA2 and U46619 induced sustained aggregation. Blocking the IP receptor switched F2-TXA2-mediated reversible aggregation into sustained aggregation. Further BRET studies confirmed F2-TXA2-mediated IP receptor activation. F2-TXA2 rapidly and potently stimulated platelet TP receptor-mediated protein kinase C/P-pleckstrin, whereas IP-mediated protein kinase A/P-vasodilator-stimulated phosphoprotein was more delayed. CONCLUSION AND IMPLICATIONS: F-TXA2 is a close analogue to TXA2 used as a selective tool for TP receptor platelet activation. In contrast, F2-TXA2 acts on both TP and IP receptors differently over time, resulting in an initial wave of TP receptor-mediated platelet aggregation followed by IP receptor-induced reversibility of aggregation. This study reveals the potential difference in the temporal aspects of stimulatory and inhibitory pathways involved in platelet activation.

2.
Br J Pharmacol ; 181(1): 21-35, 2024 01.
Article in English | MEDLINE | ID: mdl-37530222

ABSTRACT

BACKGROUND AND PURPOSE: Ticagrelor is labelled as a reversible, direct-acting platelet P2Y12 receptor (P2Y12 R) antagonist that is indicated clinically for the prevention of thrombotic events in patients with acute coronary syndrome (ACS). As with many antiplatelet drugs, ticagrelor therapy increases bleeding risk in patients, which may require platelet transfusion in emergency situations. The aim of this study was to further examine the reversibility of ticagrelor at the P2Y12 R. EXPERIMENTAL APPROACH: Studies were performed in human platelets, with P2Y12 R-stimulated GTPase activity and platelet aggregation assessed. Cell-based bioluminescence resonance energy transfer (BRET) assays were undertaken to assess G protein-subunit activation downstream of P2Y12 R activation. KEY RESULTS: Initial studies revealed that a range of P2Y12 R ligands, including ticagrelor, displayed inverse agonist activity at P2Y12 R. Only ticagrelor was resistant to washout and, in human platelet and cell-based assays, washing failed to reverse ticagrelor-dependent inhibition of ADP-stimulated P2Y12 R function. The P2Y12 R agonist 2MeSADP, which was also resistant to washout, was able to effectively compete with ticagrelor. In silico docking revealed that ticagrelor and 2MeSADP penetrated more deeply into the orthosteric binding pocket of the P2Y12 R than other P2Y12 R ligands. CONCLUSION AND IMPLICATIONS: Ticagrelor binding to P2Y12 R is prolonged and more akin to that of an irreversible antagonist, especially versus the endogenous P2Y12 R agonist ADP. This study highlights the potential clinical need for novel ticagrelor reversal strategies in patients with spontaneous major bleeding, and for bleeding associated with urgent invasive procedures.


Subject(s)
Acute Coronary Syndrome , Diphosphates , Humans , Ticagrelor/pharmacology , Ticagrelor/metabolism , Ticagrelor/therapeutic use , Diphosphates/metabolism , Diphosphates/pharmacology , Diphosphates/therapeutic use , Adenosine/pharmacology , Drug Inverse Agonism , Purinergic P2Y Receptor Antagonists/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Adenosine Diphosphate/pharmacology , Adenosine Diphosphate/metabolism , Blood Platelets , Acute Coronary Syndrome/drug therapy , Acute Coronary Syndrome/complications , Receptors, Purinergic P2Y12/metabolism
3.
J Thromb Haemost ; 21(5): 1307-1321, 2023 05.
Article in English | MEDLINE | ID: mdl-36716966

ABSTRACT

BACKGROUND: Patients with COVID-19 are at increased risk of thrombosis, which is associated with altered platelet function and coagulopathy, contributing to excess mortality. OBJECTIVES: To characterize the mechanism of altered platelet function in COVID-19 patients. METHODS: The platelet proteome, platelet functional responses, and platelet-neutrophil aggregates were compared between patients hospitalized with COVID-19 and healthy control subjects using tandem mass tag proteomic analysis, Western blotting, and flow cytometry. RESULTS: COVID-19 patients showed a different profile of platelet protein expression (858 altered of the 5773 quantified). Levels of COVID-19 plasma markers were enhanced in the platelets of COVID-19 patients. Gene ontology pathway analysis demonstrated that the levels of granule secretory proteins were raised, whereas those of platelet activation proteins, such as the thrombopoietin receptor and protein kinase Cα, were lowered. Basally, platelets of COVID-19 patients showed enhanced phosphatidylserine exposure, with unaltered integrin αIIbß3 activation and P-selectin expression. Agonist-stimulated integrin αIIbß3 activation and phosphatidylserine exposure, but not P-selectin expression, were decreased in COVID-19 patients. COVID-19 patients had high levels of platelet-neutrophil aggregates, even under basal conditions, compared to controls. This association was disrupted by blocking P-selectin, demonstrating that platelet P-selectin is critical for the interaction. CONCLUSIONS: Overall, our data suggest the presence of 2 platelet populations in patients with COVID-19: one of circulating platelets with an altered proteome and reduced functional responses and another of P-selectin-expressing neutrophil-associated platelets. Platelet-driven thromboinflammation may therefore be one of the key factors enhancing the risk of thrombosis in COVID-19 patients.


Subject(s)
COVID-19 , Thrombosis , Humans , Proteome/metabolism , COVID-19/complications , Proteomics , Phosphatidylserines/metabolism , Inflammation/metabolism , Thrombosis/etiology , Blood Platelets/metabolism , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Platelet Activation , Selectins/metabolism
4.
Blood Adv ; 5(7): 1884-1898, 2021 04 13.
Article in English | MEDLINE | ID: mdl-33792632

ABSTRACT

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.


Subject(s)
Antigens, CD , Bone Marrow Stromal Antigen 2 , Animals , Antigens, CD/genetics , Blood Platelets , Cell Line , GPI-Linked Proteins/genetics , Mice
6.
Haematologica ; 106(7): 1968-1978, 2021 07 01.
Article in English | MEDLINE | ID: mdl-32467143

ABSTRACT

Pim kinases are upregulated in several forms of cancer, contributing to cell survival and tumour development, but their role in platelet function and thrombotic disease has not been explored. We report for the first time that Pim-1 is expressed in human and mouse platelets. Genetic deletion or pharmacological inhibition of Pim kinase results in reduced thrombus formation but is not associated with impaired haemostasis. Attenuation of thrombus formation was found to be due to inhibition of the thromboxane A2 receptor as effects on platelet function was non-additive to inhibition caused by the cyclooxygenase inhibitor indomethacin or thromboxane A2 receptor antagonist GR32191. Treatment with Pim kinase inhibitors caused reduced surface expression of the thromboxane A2 receptor and resulted in reduced responses to thromboxane A2 receptor agonists, indicating a role for Pim kinase in the regulation of thromboxane A2 receptor function. Our research identifies a novel, Pim kinase dependent regulatory mechanism for the thromboxane A2 receptor and represents a new targeting strategy that is independent of COX-1 inhibition or direct antagonism of the thromboxane A2 receptor that whilst attenuating thrombosis does not increase bleeding.


Subject(s)
Receptors, Thromboxane A2, Prostaglandin H2 , Thrombosis , Blood Platelets , Humans , Platelet Aggregation , Proto-Oncogene Proteins c-pim-1/genetics , Receptors, Thromboxane A2, Prostaglandin H2/genetics , Thrombosis/drug therapy
7.
J Am Heart Assoc ; 9(24): e016495, 2020 12 15.
Article in English | MEDLINE | ID: mdl-33305660

ABSTRACT

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 A2 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.


Subject(s)
Acute Coronary Syndrome/drug therapy , Drug Therapy, Combination/adverse effects , Percutaneous Coronary Intervention/adverse effects , Purinergic P2Y Receptor Antagonists/pharmacology , Ticagrelor/pharmacology , Acute Coronary Syndrome/blood , Aged , Arachidonic Acid/blood , Aspirin/therapeutic use , Drug Therapy, Combination/methods , Dual Anti-Platelet Therapy/adverse effects , Dual Anti-Platelet Therapy/methods , Female , Humans , Male , Middle Aged , Peptide Fragments/drug effects , Platelet Aggregation/drug effects , Platelet Aggregation Inhibitors/therapeutic use , Platelet Function Tests/methods , Purinergic P2Y Receptor Antagonists/administration & dosage , Purinergic P2Y Receptor Antagonists/therapeutic use , Receptors, Thromboxane A2, Prostaglandin H2/agonists , Ticagrelor/administration & dosage , Ticagrelor/therapeutic use
8.
Platelets ; 31(5): 641-645, 2020 Jul 03.
Article in English | MEDLINE | ID: mdl-31684789

ABSTRACT

The principal demonstrated role of the nonvisual arrestins in vivo is to limit G protein-coupled receptor (GPCR) signaling. Nonetheless, a direct demonstration of this fundamental ability in platelets remains lacking, despite the prominent role played by GPCRs in platelet activation. This paper describes the basic characterization of the activatory responses of platelets from mice lacking arrestin-3 (arr3-/-), revealing pleiotropic roles dependent on GPCR ligand. Functionally, arrestin-3 acts as a brake on platelet aggregation regardless of ligand tested. Downstream of P2Y receptors, arr3-/- mice show increased secretion and integrin activation mirrored by enhanced intracellular calcium signaling and global PKC-dependent phosphorylation. Furthermore, P2Y12 receptor (P2Y12R) activity as assessed by ADP-mediated reduction of VASP phosphorylation is enhanced in arr3-/-mice. Downstream of PAR receptors there are similar increases in secretion and integrin activation in arr3-/- mice, together with enhanced PKC activity. Last, in arr3-/- mice the TP receptor displays unaltered PKC activity but markedly reduced calcium responses, which together with the kinetics of the aggregation response suggested a unique positive regulatory role for arrestin-3 in TP signaling. Overall, this paper reveals pleiotropic roles for arrestin-3 dependent on GPCR ligand describing for the first time a negative regulatory function for arrestin-3 in platelets.


Subject(s)
Arrestins/metabolism , Blood Platelets/metabolism , Receptors, G-Protein-Coupled/metabolism , Animals , Blood Platelets/cytology , Humans , Mice
9.
Platelets ; 29(7): 739-742, 2018 Nov.
Article in English | MEDLINE | ID: mdl-30089223

ABSTRACT

Platelet activity is regulated by a number of surface expressed G protein-coupled receptors (GPCRs) including the α isoform of the thromboxane receptor (TPα receptor). With the advance of genomic technologies, there has been a substantial increase in the identification of naturally occurring rare GPCR variants including in the TBXA2R gene, which encodes the TPα receptor. The study of patients with naturally occurring variants within TBXA2R associated with bleeding and abnormal TPα receptor function has provided a powerful insight in defining the critical role of TPα in thrombus formation. This review will highlight how the identification of these function-disrupting variants of the platelet TPα has contributed important structure-function information about these GPCRs. Further we discuss the potential implications these findings have for understanding the molecular basis of mild platelet based bleeding disorders.


Subject(s)
Genetic Association Studies , Genetic Predisposition to Disease , Genetic Variation , Hemorrhage/blood , Hemorrhage/genetics , Receptors, Thromboxane A2, Prostaglandin H2/genetics , Alleles , Genotype , Hemorrhage/diagnosis , Humans , Phenotype
10.
Mol Cell Endocrinol ; 449: 74-81, 2017 07 05.
Article in English | MEDLINE | ID: mdl-28212842

ABSTRACT

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 (P2Y1 and P2Y12), 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 P2Y12 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.


Subject(s)
Blood Platelets/metabolism , Receptors, Purinergic P2Y12/metabolism , Animals , Disease , Genetic Variation , Humans , Models, Biological , Platelet Activation , Receptors, Purinergic P2Y12/chemistry , Receptors, Purinergic P2Y12/genetics
11.
Biochem Pharmacol ; 124: 43-56, 2017 Jan 15.
Article in English | MEDLINE | ID: mdl-27845050

ABSTRACT

Thromboxane A2 is a potent mediator of inflammation and platelet aggregation exerting its effects through the activation of a G protein-coupled receptor (GPCR), termed TP. Although the existence of dimers/oligomers in Class A GPCRs is widely accepted, their functional significance still remains controversial. Recently, we have shown that TPα and TPß homo-/hetero-dimers interact through an interface of residues in transmembrane domain 1 (TM1) whose disruption impairs dimer formation. Here, biochemical and pharmacological characterization of this dimer deficient mutant (DDM) in living cells indicates a significant impairment in its response to agonists. Interestingly, two single loss-of-function TPα variants, namely W29C and N42S recently identified in two heterozygous patients affected by bleeding disorders, match some of the residues mutated in our DDM. These two naturally occurring variants display a reduced potency to TP agonists and are characterized by impaired dimer formation in transfected HEK-293T cells. These findings provide proofs that lack of homo-dimer formation is a crucial process for reduced TPα function in vivo, and might represent one molecular mechanism through which platelet TPα receptor dysfunction affects the patient(s) carrying these mutations.


Subject(s)
Blood Platelets/physiology , Receptors, Thromboxane/metabolism , Signal Transduction , Dimerization , Dose-Response Relationship, Drug , HEK293 Cells , Humans , Ligands , Mutation , Receptors, Thromboxane/agonists , Receptors, Thromboxane/antagonists & inhibitors , Receptors, Thromboxane/genetics
12.
PLoS One ; 11(10): e0163006, 2016.
Article in English | MEDLINE | ID: mdl-27716777

ABSTRACT

Thioredoxin (Trx) is an oxidoreductase with important physiological function. Imbalances in the NADPH/thioredoxin reductase/thioredoxin system are associated with a number of pathologies, particularly cancer, and a number of clinical trials for thioredoxin and thioredoxin reductase inhibitors have been carried out or are underway. Due to the emerging role and importance of oxidoreductases for haemostasis and the current interest in developing inhibitors for clinical use, we thought it pertinent to assess whether inhibition of the NADPH/thioredoxin reductase/thioredoxin system affects platelet function and thrombosis. We used small molecule inhibitors of Trx (PMX 464 and PX-12) to determine whether Trx activity influences platelet function, as well as an unbiased proteomics approach to identify potential Trx substrates on the surface of platelets that might contribute to platelet reactivity and function. Using LC-MS/MS we found that PMX 464 and PX-12 affected the oxidation state of thiols in a number of cell surface proteins. Key surface receptors for platelet adhesion and activation were affected, including the collagen receptor GPVI and the von Willebrand factor receptor, GPIb. To experimentally validate these findings we assessed platelet function in the presence of PMX 464, PX-12, and rutin (a selective inhibitor of the related protein disulphide isomerase). In agreement with the proteomics data, small molecule inhibitors of thioredoxin selectively inhibited GPVI-mediated platelet activation, and attenuated ristocetin-induced GPIb-vWF-mediated platelet agglutination, thus validating the findings of the proteomics study. These data reveal a novel role for thioredoxin in regulating platelet reactivity via proteins required for early platelet responses at sites of vessel injury (GPVI and GPIb). This work also highlights a potential opportunity for repurposing of PMX 464 and PX-12 as antiplatelet agents.


Subject(s)
Blood Platelets/drug effects , Platelet Aggregation Inhibitors/pharmacology , Thioredoxins/pharmacology , Thrombosis/drug therapy , Benzothiazoles/pharmacology , Blood Coagulation Tests/methods , Blood Platelets/metabolism , Disulfides/pharmacology , Humans , Hydroquinones/pharmacology , Imidazoles/pharmacology , Platelet Activation/drug effects , Platelet Adhesiveness/drug effects , Platelet Aggregation/drug effects , Platelet Function Tests/methods , Platelet Membrane Glycoproteins/metabolism , Receptors, Collagen/metabolism , Ristocetin/pharmacology , Thrombosis/metabolism , von Willebrand Factor/metabolism
13.
Blood ; 128(23): 2717-2728, 2016 12 08.
Article in English | MEDLINE | ID: mdl-27694321

ABSTRACT

Ticagrelor is a potent antagonist of the P2Y12 receptor (P2Y12R) 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 Ca2+ release in washed platelets vs other P2Y12R antagonists. This additional effect of ticagrelor beyond P2Y12R 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 Gs-coupled adenosine A2A 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 P2Y12R signaling, and this was validated in 1321N1 cells stably transfected with human P2Y12R. In these cells, ticagrelor blocked the constitutive agonist-independent activity of the P2Y12R, limiting basal Gi-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 Ca2+ 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 P2Y12R that contribute to its effective inhibition of platelet activation.


Subject(s)
Adenosine/analogs & derivatives , Blood Platelets/metabolism , Equilibrative Nucleoside Transporter 1/antagonists & inhibitors , Platelet Activation/drug effects , Receptors, Purinergic P2Y12/metabolism , Adenosine/pharmacology , Adenosine Diphosphate/pharmacology , Blood Platelets/cytology , Calcium/metabolism , Calcium Signaling/drug effects , Cell Adhesion Molecules/metabolism , Colforsin/pharmacology , Cyclic AMP/metabolism , Equilibrative Nucleoside Transporter 1/metabolism , Female , Humans , Male , Microfilament Proteins/metabolism , Phosphoproteins/metabolism , Phosphorylation/drug effects , Ticagrelor
14.
Haematologica ; 101(10): 1170-1179, 2016 10.
Article in English | MEDLINE | ID: mdl-27479822

ABSTRACT

Inherited thrombocytopenias are a heterogeneous group of disorders characterized by abnormally low platelet counts which can be associated with abnormal bleeding. Next-generation sequencing has previously been employed in these disorders for the confirmation of suspected genetic abnormalities, and more recently in the discovery of novel disease-causing genes. However its full potential has not yet been exploited. Over the past 6 years we have sequenced the exomes from 55 patients, including 37 index cases and 18 additional family members, all of whom were recruited to the UK Genotyping and Phenotyping of Platelets study. All patients had inherited or sustained thrombocytopenia of unknown etiology with platelet counts varying from 11×109/L to 186×109/L. Of the 51 patients phenotypically tested, 37 (73%), had an additional secondary qualitative platelet defect. Using whole exome sequencing analysis we have identified "pathogenic" or "likely pathogenic" variants in 46% (17/37) of our index patients with thrombocytopenia. In addition, we report variants of uncertain significance in 12 index cases, including novel candidate genetic variants in previously unreported genes in four index cases. These results demonstrate that whole exome sequencing is an efficient method for elucidating potential pathogenic genetic variants in inherited thrombocytopenia. Whole exome sequencing also has the added benefit of discovering potentially pathogenic genetic variants for further study in novel genes not previously implicated in inherited thrombocytopenia.


Subject(s)
Exome/genetics , Genetic Variation , High-Throughput Nucleotide Sequencing , Thrombocytopenia/genetics , Blood Platelets/pathology , Genetic Predisposition to Disease , Humans , Mutation, Missense , Platelet Count
15.
Arterioscler Thromb Vasc Biol ; 36(5): 952-60, 2016 05.
Article in English | MEDLINE | ID: mdl-26966273

ABSTRACT

OBJECTIVE: Protease-activated receptor 4 (PAR4) is a key regulator of platelet reactivity and is encoded by F2RL3, which has abundant rare missense variants. We aimed to provide proof of principle that rare F2LR3 variants potentially affect platelet reactivity and responsiveness to PAR1 antagonist drugs and to explore underlying molecular mechanisms. APPROACH AND RESULTS: We identified 6 rare F2RL3 missense variants in 236 cardiac patients, of which the variant causing a tyrosine 157 to cysteine substitution (Y157C) was predicted computationally to have the greatest effect on PAR4 structure. Y157C platelets from 3 cases showed reduced responses to PAR4-activating peptide and to α-thrombin compared with controls, but no reduction in responses to PAR1-activating peptide. Pretreatment with the PAR1 antagonist vorapaxar caused lower residual α-thrombin responses in Y157C platelets than in controls, indicating greater platelet inhibition. HEK293 cells transfected with a PAR4 Y157C expression construct had reduced PAR4 functional responses, unchanged total PAR4 expression but reduced surface expression. PAR4 Y157C was partially retained in the endoplasmic reticulum and displayed an expression pattern consistent with defective N-glycosylation. Mutagenesis of Y322, which is the putative hydrogen bond partner of Y157, also reduced PAR4 surface expression in HEK293 cells. CONCLUSIONS: Reduced PAR4 responses associated with Y157C result from aberrant anterograde surface receptor trafficking, in part, because of disrupted intramolecular hydrogen bonding. Characterization of PAR4 Y157C establishes that rare F2RL3 variants have the potential to markedly alter platelet PAR4 reactivity particularly after exposure to therapeutic PAR1 antagonists.


Subject(s)
Blood Platelets/metabolism , Platelet Activation , Receptors, Thrombin/metabolism , Aged , Blood Platelets/drug effects , Case-Control Studies , Computer Simulation , Dose-Response Relationship, Drug , Endoplasmic Reticulum/metabolism , England , Female , Genotype , Glycosylation , HEK293 Cells , Humans , Hydrogen Bonding , Lactones/pharmacology , Male , Models, Molecular , Mutation, Missense , Peptides/pharmacology , Phenotype , Platelet Activation/drug effects , Platelet Aggregation , Platelet Aggregation Inhibitors/pharmacology , Polymorphism, Single Nucleotide , Protein Conformation , Protein Transport , Pyridines/pharmacology , Receptor, PAR-1/drug effects , Receptor, PAR-1/metabolism , Receptors, Thrombin/chemistry , Receptors, Thrombin/drug effects , Receptors, Thrombin/genetics , Structure-Activity Relationship , Thrombin/pharmacology , Transfection
16.
PLoS One ; 10(12): e0144984, 2015.
Article in English | MEDLINE | ID: mdl-26672598

ABSTRACT

BACKGROUND: Rapid coronary recanalization following ST-elevation myocardial infarction (STEMI) requires effective anti-platelet and anti-thrombotic therapies. This study tested the impact of door to end of procedure ('door-to-end') time and baseline platelet activity on platelet inhibition within 24hours post-STEMI. METHODS AND FINDINGS: 108 patients, treated with prasugrel and procedural bivalirudin, underwent Multiplate® platelet function testing at baseline, 0, 1, 2 and 24hours post-procedure. Major adverse cardiac events (MACE), bleeding and stent thrombosis (ST) were recorded. Baseline ADP activity was high (88.3U [71.8-109.0]), procedural time and consequently bivalirudin infusion duration were short (median door-to-end time 55minutes [40-70] and infusion duration 30minutes [20-42]). Baseline ADP was observed to influence all subsequent measurements of ADP activity, whereas door-to-end time only influenced ADP immediately post-procedure. High residual platelet reactivity (HRPR ADP>46.8U) was observed in 75% of patients immediately post-procedure and persisted in 24% of patients at 2hours. Five patients suffered in-hospital MACE (4.6%). Acute ST occurred in 4 patients, all were <120mins post-procedure and had HRPR. No significant bleeding was observed. In a post-hoc analysis, pre-procedural morphine use was associated with significantly higher ADP activity following intervention. CONCLUSIONS: Baseline platelet function, time to STEMI treatment and opiate use all significantly influence immediate post-procedural platelet activity.


Subject(s)
Myocardial Infarction/therapy , Percutaneous Coronary Intervention , Platelet Aggregation Inhibitors/therapeutic use , Platelet Function Tests , Point-of-Care Systems , Aged , Blood Platelets/drug effects , Blood Platelets/metabolism , Coronary Angiography , Female , Humans , Male , Middle Aged , Myocardial Infarction/blood , Myocardial Infarction/diagnosis , Myocardial Infarction/etiology , Percutaneous Coronary Intervention/adverse effects , Platelet Activation/drug effects , Platelet Aggregation Inhibitors/pharmacology , Time Factors , Treatment Outcome
17.
PLoS One ; 10(12): e0143913, 2015.
Article in English | MEDLINE | ID: mdl-26630678

ABSTRACT

The clinical expression of type 1 von Willebrand disease may be modified by co-inheritance of other mild bleeding diatheses. We previously showed that mutations in the platelet P2Y12 ADP receptor gene (P2RY12) could contribute to the bleeding phenotype in patients with type 1 von Willebrand disease. Here we investigated whether variations in platelet G protein-coupled receptor genes other than P2RY12 also contributed to the bleeding phenotype. Platelet G protein-coupled receptor genes P2RY1, F2R, F2RL3, TBXA2R and PTGIR were sequenced in 146 index cases with type 1 von Willebrand disease and the potential effects of identified single nucleotide variations were assessed using in silico methods and heterologous expression analysis. Seven heterozygous single nucleotide variations were identified in 8 index cases. Two single nucleotide variations were detected in F2R; a novel c.-67G>C transversion which reduced F2R transcriptional activity and a rare c.1063C>T transition predicting a p.L355F substitution which did not interfere with PAR1 expression or signalling. Two synonymous single nucleotide variations were identified in F2RL3 (c.402C>G, p.A134 =; c.1029 G>C p.V343 =), both of which introduced less commonly used codons and were predicted to be deleterious, though neither of them affected PAR4 receptor expression. A third single nucleotide variation in F2RL3 (c.65 C>A; p.T22N) was co-inherited with a synonymous single nucleotide variation in TBXA2R (c.6680 C>T, p.S218 =). Expression and signalling of the p.T22N PAR4 variant was similar to wild-type, while the TBXA2R variation introduced a cryptic splice site that was predicted to cause premature termination of protein translation. The enrichment of single nucleotide variations in G protein-coupled receptor genes among type 1 von Willebrand disease patients supports the view of type 1 von Willebrand disease as a polygenic disorder.


Subject(s)
Blood Platelets/metabolism , Receptors, G-Protein-Coupled/genetics , von Willebrand Diseases/genetics , von Willebrand Factor/genetics , 5' Untranslated Regions , Animals , Base Sequence , HEK293 Cells , Hemorrhage/physiopathology , Humans , Molecular Sequence Data , Polymorphism, Single Nucleotide , Sequence Homology, Nucleic Acid , Transcription, Genetic
18.
Thromb Res ; 136(4): 818-24, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26297398

ABSTRACT

INTRODUCTION: Multiple electrode aggregometry (MEA) improves prediction of thrombosis and bleeding in cardiac patients. However, the causes of inter-individual variation in MEA results are incompletely understood. We explore whether low MEA results are associated with platelet G-protein coupled receptor (GPCR) gene variants. METHODS: The effects of P2Y12 receptor (P2Y12), thromboxane A2 receptor (TPα) and protease-activated receptor 1 (PAR1) dysfunction on the MEA ADP-test, ASPI-test and TRAP-test were determined using receptor antagonists. Cardiac surgery patients with pre-operative MEA results suggesting GPCR dysfunction were selected for P2Y12 (P2RY12), TPα (TBXA2R) and PAR1 (F2R) sequencing. RESULTS: In control blood samples, P2Y12, TPα or PAR1 antagonists markedly reduced ADP-test, ASPI-test and TRAP-test results respectively. In the 636 patients from a cohort of 2388 cardiac surgery patients who were not receiving aspirin or a P2Y12 blocker, the median ADP-test result was 75.1 U (range 4.8-153.2), ASPI-test 83.7 U (1.4-157.3) and TRAP-test 117.7 U (2.4-194.1), indicating a broad range of results unexplained by anti-platelet drugs. In 238 consenting patients with unexplained low MEA results, three P2RY12 variants occurred in 70/107 (65%) with suspected P2Y12 dysfunction and four TBXA2R variants occurred in 19/22 (86%) with suspected TPα dysfunction although the later group was too small to draw meaningful conclusions about variant frequency. All the variants were synonymous and unlikely to cause GPCR dysfunction. There were no F2R variants in the 109 cases with suspected PAR1 dysfunction. CONCLUSION: MEA results suggesting isolated platelet GPCR dysfunction were common in cardiac surgery patients, but were not associated with non-synonymous variants in P2RY12 or F2R.


Subject(s)
Blood Platelets/metabolism , Platelet Aggregation/genetics , Platelet Function Tests/methods , Receptors, G-Protein-Coupled/genetics , Female , Humans , Male , Receptors, G-Protein-Coupled/biosynthesis , Signal Transduction
19.
Thromb Haemost ; 113(4): 826-37, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25567036

ABSTRACT

Platelet responses to activating agonists are influenced by common population variants within or near G protein-coupled receptor (GPCR) genes that affect receptor activity. However, the impact of rare GPCR gene variants is unknown. We describe the rare single nucleotide variants (SNVs) in the coding and splice regions of 18 GPCR genes in 7,595 exomes from the 1,000-genomes and Exome Sequencing Project databases and in 31 cases with inherited platelet function disorders (IPFDs). In the population databases, the GPCR gene target regions contained 740 SNVs (318 synonymous, 410 missense, 7 stop gain and 6 splice region) of which 70 % had global minor allele frequency (MAF) < 0.05 %. Functional annotation using six computational algorithms, experimental evidence and structural data identified 156/740 (21 %) SNVs as potentially damaging to GPCR function, most commonly in regions encoding the transmembrane and C-terminal intracellular receptor domains. In 31 index cases with IPFDs (Gi-pathway defect n=15; secretion defect n=11; thromboxane pathway defect n=3 and complex defect n=2) there were 256 SNVs in the target regions of 15 stimulatory platelet GPCRs (34 unique; 12 with MAF< 1 % and 22 with MAF≥ 1 %). These included rare variants predicting R122H, P258T and V207A substitutions in the P2Y12 receptor that were annotated as potentially damaging, but only partially explained the platelet function defects in each case. Our data highlight that potentially damaging variants in platelet GPCR genes have low individual frequencies, but are collectively abundant in the population. Potentially damaging variants are also present in pedigrees with IPFDs and may contribute to complex laboratory phenotypes.


Subject(s)
Blood Platelet Disorders/blood , Blood Platelet Disorders/genetics , Blood Platelets/metabolism , Polymorphism, Single Nucleotide , Receptors, G-Protein-Coupled/blood , Receptors, G-Protein-Coupled/genetics , Computational Biology , Databases, Genetic , Exome , Female , Gene Frequency , Genetic Predisposition to Disease , HEK293 Cells , Heredity , Humans , Male , Pedigree , Phenotype , Platelet Function Tests , Receptors, Purinergic P2Y12/blood , Receptors, Purinergic P2Y12/genetics , Transfection
20.
Psychopharmacology (Berl) ; 232(1): 305-14, 2015 Jan.
Article in English | MEDLINE | ID: mdl-24973897

ABSTRACT

RATIONALE: The novel opioid receptor antagonist, GSK1421498, has been shown to attenuate reward-driven compulsive behaviours, such as stimulant drug seeking or binge eating, in animals and humans. Here, we report new data on the receptor pharmacology of GSK121498, in comparison to naltrexone, naloxone, 6-ß-naltrexol and nalmefene. OBJECTIVES: To determine whether the novel opioid antagonist, GSK1521498, is an orthosteric or allosteric antagonist at the µ opioid receptor (MOPr) and whether it has neutral antagonist or inverse agonist properties. METHODS: A combination of radioligand binding assays and [(35)S]GTPγS binding assays was employed. RESULTS: GSK1521498 completely displaced [(3)H]naloxone binding to MOPr and did not alter the rate of [(3)H]naloxone dissociation from MOPr observations compatible with it binding to the orthosteric site on MOPr. GSK1521498 exhibited inverse agonism when MOPr was overexpressed but not when the level of MOPr expression was low. In parallel studies under conditions of high receptor expression density, naloxone, naltrexone, 6-ß-naltrexol and nalmefene exhibited partial agonism, not inverse agonism as has been reported previously for naloxone and naltrexone. In brain tissue from mice receiving a prolonged morphine pre-treatment, GSK1521498 exhibited slight inverse agonism. CONCLUSIONS: Differences between GSK1521498 and naltrexone in their effects on compulsive reward seeking are arguably linked to the more selective and complete MOPr antagonism of GSK1521498 versus the partial MOPr agonism of naltrexone. GSK1521498 is also pharmacologically differentiated by its inverse agonist efficacy at high levels of MOPr expression, but this may be less likely to contribute to behavioural differentiation at patho-physiological levels of expression.


Subject(s)
Compulsive Behavior/metabolism , Indans/pharmacology , Receptors, Opioid, mu/antagonists & inhibitors , Reward , Triazoles/pharmacology , Analgesics, Opioid/metabolism , Analgesics, Opioid/pharmacology , Analgesics, Opioid/therapeutic use , Animals , CHO Cells , Compulsive Behavior/drug therapy , Cricetinae , Cricetulus , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , HEK293 Cells , Humans , Indans/metabolism , Indans/therapeutic use , Ligands , Male , Mice , Morphine/metabolism , Morphine/pharmacology , Morphine/therapeutic use , Naloxone/metabolism , Naloxone/pharmacology , Naloxone/therapeutic use , Naltrexone/analogs & derivatives , Naltrexone/metabolism , Naltrexone/pharmacology , Naltrexone/therapeutic use , Narcotic Antagonists/metabolism , Narcotic Antagonists/pharmacology , Narcotic Antagonists/therapeutic use , Protein Binding/physiology , Receptors, Opioid, mu/metabolism , Treatment Outcome , Triazoles/metabolism , Triazoles/therapeutic use
SELECTION OF CITATIONS
SEARCH DETAIL
...