Profiling oxylipins released from human platelets activated through the GPVI collagen receptor.

In addition to haemostasis, platelets are involved in pathological processes, often driven by material released upon activation. Interaction between collagen and glycoprotein VI (GPVI) is a primary platelet stimulus that liberates arachidonic acid and linoleic acid from membrane phospholipids. These are oxidised by cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12-LOX) to eicosanoids and other oxylipins with various biological properties. Using liquid chromatography-tandem mass spectrometry we found that GPVI-stimulated platelets released significant levels of ten oxylipins; the well documented TxA2 and 12-HETE, PGD2 and PGE2, as well as 8-, 9-, 11-, and 15-HETE, 9- and 13-HODE.1 Levels of oxylipins released from washed platelets mirrored those from platelets stimulated in the presence of plasma, indicating generation from intracellular, rather than exogenous AA/LA. Inhibition of COX-1 with aspirin, as expected, completely abolished production of TxA2 and PGD/E2, but also significantly inhibited the release of 11-HETE (89 ± 3%) and 9-HODE (74 ± 6%), and reduced 15-HETE and 13-HODE by ∼33 %. Inhibition of 12-LOX by either esculetin or ML355 inhibited the release of all oxylipins apart from 15-HETE. These findings suggest routes to modify the production of bioactive molecules released by activated platelets.

Does hsa-miR-223-3p from platelet-derived extracellular vesicles regulate tissue factor expression in monocytic cells?

Extracellular vesicles (EVs) released from activated platelets contain microRNAs, the most abundant of which is hsa-miR-223-3p. Endogenous hsa-miR-223-3p suppresses the expression of tissue factor (TF), the initiator of the extrinsic coagulation pathway, in endothelial cells. Monocytes can be induced to express TF to enhance coagulation, but the role of hsa-miR-223-3p in regulating monocyte TF remains unknown. This study examined whether hsa-miR-223-3p from platelet-derived EVs (pdEVs) affects TF expression in monocytes. THP-1 cells, differentiated into a monocyte-like phenotype with 1α,25-dihydroxyvitaminD3, were transfected with hsa-miR-223-3p mimic or control microRNA. Alternatively, THP-1 cells were incubated with pdEVs from PAR1-agonist peptide activated-platelets, as platelet releasate, or pdEVs isolated by ultracentrifugation. Transfection with hsa-miR-223-3p mimic resulted in significant reductions in TF protein, determined by western blotting and flow cytometry and reduced procoagulant activity, measured by a TF-specific factor Xa generation assay, compared to cells transfected with control microRNA. This reduction was reversed by co-transfection with hsa-miR-223-3p inhibitor, AntagomiR-223. Incubation of THP-1 cells with pdEVs also decreased TF expression; however, this was not reversed by AntagomiR-223. Taken together, monocyte TF expression is downregulated by hsa-miR-223-3p, but when transferred via pdEVs the effect was not reversed with Antagomir-223, suggesting other pdEV components may contribute to TF regulation.Abbreviations: Tissue factor (TF), Factor VII (FVII), activated Factor VII (FVIIa), Factor X (FX), activated Factor X (FXa), extracellular vesicles (EVs), microvesicles (MVs), platelet-derived extracellular vesicles (pdEVs), protease-activated receptor 1 agonist peptide (PAR1-AP), lipopolysaccharide (LPS), P-selectin glycoprotein ligand-1 (PSGL-1), Tris-Buffered Saline Tween (TBST), room temperature (RT)[Figure: see text].

Inorganic Phosphate (Pi) Signaling in Endothelial Cells: A Molecular Basis for Generation of Endothelial Microvesicles in Uraemic Cardiovascular Disease.

Hyperphosphataemia increases cardiovascular mortality in patients with kidney disease. Direct effects of high inorganic phosphate (Pi) concentrations have previously been demonstrated on endothelial cells (ECs), including generation of procoagulant endothelial microvesicles (MVs). However, no mechanism directly sensing elevated intracellular Pi has ever been described in mammalian cells. Here, we investigated the hypothesis that direct inhibition by Pi of the phosphoprotein phosphatase PP2A fulfils this sensing role in ECs, culminating in cytoskeleton disruption and MV generation. ECs were treated with control (1 mM [Pi]) vs. high (2.5 mM [Pi]), a condition that drives actin stress fibre depletion and MV generation demonstrated by confocal microscopy of F-actin and NanoSight Nanoparticle tracking, respectively. Immuno-blotting demonstrated that high Pi increased p-Src, p-PP2A-C and p-DAPK-1 and decreased p-TPM-3. Pi at 100 µM directly inhibited PP2A catalytic activity. Inhibition of PP2A enhanced inhibitory phosphorylation of DAPK-1, leading to hypophosphorylation of Tropomyosin-3 at S284 and MV generation. p-Src is known to perform inhibitory phosphorylation on DAPK-1 but also on PP2A-C. However, PP2A-C can itself dephosphorylate (and therefore inhibit) p-Src. The direct inhibition of PP2A-C by Pi is, therefore, amplified by the feedback loop between PP2A-C and p-Src, resulting in further PP2A-C inhibition. These data demonstrated that PP2A/Src acts as a potent sensor and amplifier of Pi signals which can further signal through DAPK-1/Tropomyosin-3 to generate cytoskeleton disruption and generation of potentially pathological MVs.

Evidence for shear-mediated Ca2+ entry through mechanosensitive cation channels in human platelets and a megakaryocytic cell line.

The role of mechanosensitive (MS) Ca2+-permeable ion channels in platelets is unclear, despite the importance of shear stress in platelet function and life-threatening thrombus formation. We therefore sought to investigate the expression and functional relevance of MS channels in human platelets. The effect of shear stress on Ca2+ entry in human platelets and Meg-01 megakaryocytic cells loaded with Fluo-3 was examined by confocal microscopy. Cells were attached to glass coverslips within flow chambers that allowed applications of physiological and pathological shear stress. Arterial shear (1002.6 s-1) induced a sustained increase in [Ca2+] i in Meg-01 cells and enhanced the frequency of repetitive Ca2+ transients by 80% in platelets. These Ca2+ increases were abrogated by the MS channel inhibitor Grammostola spatulata mechanotoxin 4 (GsMTx-4) or by chelation of extracellular Ca2+ Thrombus formation was studied on collagen-coated surfaces using DiOC6-stained platelets. In addition, [Ca2+] i and functional responses of washed platelet suspensions were studied with Fura-2 and light transmission aggregometry, respectively. Thrombus size was reduced 50% by GsMTx-4, independently of P2X1 receptors. In contrast, GsMTx-4 had no effect on collagen-induced aggregation or on Ca2+ influx via TRPC6 or Orai1 channels and caused only a minor inhibition of P2X1-dependent Ca2+ entry. The Piezo1 agonist, Yoda1, potentiated shear-dependent platelet Ca2+ transients by 170%. Piezo1 mRNA transcripts and protein were detected with quantitative RT-PCR and Western blotting, respectively, in both platelets and Meg-01 cells. We conclude that platelets and Meg-01 cells express the MS cation channel Piezo1, which may contribute to Ca2+ entry and thrombus formation under arterial shear.

Optical Coherence Tomography Findings in Patients With Coronary Stent Thrombosis: A Report of the PRESTIGE Consortium (Prevention of Late Stent Thrombosis by an Interdisciplinary Global European Effort).

BACKGROUND: Stent thrombosis (ST) is a serious complication following coronary stenting. Intravascular optical coherence tomography (OCT) may provide insights into mechanistic processes leading to ST. We performed a prospective, multicenter study to evaluate OCT findings in patients with ST. METHODS: Consecutive patients presenting with ST were prospectively enrolled in a registry by using a centralized telephone registration system. After angiographic confirmation of ST, OCT imaging of the culprit vessel was performed with frequency domain OCT. Clinical data were collected according to a standardized protocol. OCT acquisitions were analyzed at a core laboratory. Dominant and contributing findings were adjudicated by an imaging adjudication committee. RESULTS: Two hundred thirty-one patients presenting with ST underwent OCT imaging; 14 (6.1%) had image quality precluding further analysis. Of the remaining patients, 62 (28.6%) and 155 (71.4%) presented with early and late/very late ST, respectively. The underlying stent type was a new-generation drug-eluting stent in 50.3%. Mean reference vessel diameter was 2.9±0.6 mm and mean reference vessel area was 6.8±2.6 mm2. Stent underexpansion (stent expansion index <0.8) was observed in 44.4% of patients. The predicted average probability (95% confidence interval) that any frame had uncovered (or thrombus-covered) struts was 99.3% (96.1-99.9), 96.6% (92.4-98.5), 34.3% (15.0-60.7), and 9.6% (6.2-14.5) and malapposed struts was 21.8% (8.4-45.6), 8.5% (4.6-15.3), 6.7% (2.5-16.3), and 2.0% (1.2-3.3) for acute, subacute, late, and very late ST, respectively. The most common dominant finding adjudicated for acute ST was uncovered struts (66.7% of cases); for subacute ST, the most common dominant finding was uncovered struts (61.7%) and underexpansion (25.5%); for late ST, the most common dominant finding was uncovered struts (33.3%) and severe restenosis (19.1%); and for very late ST, the most common dominant finding was neoatherosclerosis (31.3%) and uncovered struts (20.2%). In patients presenting very late ST, uncovered stent struts were a common dominant finding in drug-eluting stents, and neoatherosclerosis was a common dominant finding in bare metal stents. CONCLUSIONS: In patients with ST, uncovered and malapposed struts were frequently observed with the incidence of both decreasing with longer time intervals between stent implantation and presentation. The most frequent dominant observation varied according to time intervals from index stenting: uncovered struts and underexpansion in acute/subacute ST and neoatherosclerosis and uncovered struts in late/very late ST.

Runs of Homozygosity: Association with Coronary Artery Disease and Gene Expression in Monocytes and Macrophages.

Runs of homozygosity (ROHs) are recognized signature of recessive inheritance. Contributions of ROHs to the genetic architecture of coronary artery disease and regulation of gene expression in cells relevant to atherosclerosis are not known. Our combined analysis of 24,320 individuals from 11 populations of white European ethnicity showed an association between coronary artery disease and both the count and the size of ROHs. Individuals with coronary artery disease had approximately 0.63 (95% CI: 0.4-0.8) excess of ROHs when compared to coronary-artery-disease-free control subjects (p = 1.49 × 10(-9)). The average total length of ROHs was approximately 1,046.92 (95% CI: 634.4-1,459.5) kb greater in individuals with coronary artery disease than control subjects (p = 6.61 × 10(-7)). None of the identified individual ROHs was associated with coronary artery disease after correction for multiple testing. However, in aggregate burden analysis, ROHs favoring increased risk of coronary artery disease were much more common than those showing the opposite direction of association with coronary artery disease (p = 2.69 × 10(-33)). Individual ROHs showed significant associations with monocyte and macrophage expression of genes in their close proximity-subjects with several individual ROHs showed significant differences in the expression of 44 mRNAs in monocytes and 17 mRNAs in macrophages when compared to subjects without those ROHs. This study provides evidence for an excess of homozygosity in coronary artery disease in outbred populations and suggest the potential biological relevance of ROHs in cells of importance to the pathogenesis of atherosclerosis.

Meta-analysis of 65,734 individuals identifies TSPAN15 and SLC44A2 as two susceptibility loci for venous thromboembolism.

Venous thromboembolism (VTE), the third leading cause of cardiovascular mortality, is a complex thrombotic disorder with environmental and genetic determinants. Although several genetic variants have been found associated with VTE, they explain a minor proportion of VTE risk in cases. We undertook a meta-analysis of genome-wide association studies (GWASs) to identify additional VTE susceptibility genes. Twelve GWASs totaling 7,507 VTE case subjects and 52,632 control subjects formed our discovery stage where 6,751,884 SNPs were tested for association with VTE. Nine loci reached the genome-wide significance level of 5 × 10(-8) including six already known to associate with VTE (ABO, F2, F5, F11, FGG, and PROCR) and three unsuspected loci. SNPs mapping to these latter were selected for replication in three independent case-control studies totaling 3,009 VTE-affected individuals and 2,586 control subjects. This strategy led to the identification and replication of two VTE-associated loci, TSPAN15 and SLC44A2, with lead risk alleles associated with odds ratio for disease of 1.31 (p = 1.67 × 10(-16)) and 1.21 (p = 2.75 × 10(-15)), respectively. The lead SNP at the TSPAN15 locus is the intronic rs78707713 and the lead SLC44A2 SNP is the non-synonymous rs2288904 previously shown to associate with transfusion-related acute lung injury. We further showed that these two variants did not associate with known hemostatic plasma markers. TSPAN15 and SLC44A2 do not belong to conventional pathways for thrombosis and have not been associated to other cardiovascular diseases nor related quantitative biomarkers. Our findings uncovered unexpected actors of VTE etiology and pave the way for novel mechanistic concepts of VTE pathophysiology.

A Comparison of Red Cell Rejuvenation versus Mechanical Washing for the Prevention of Transfusion-associated Organ Injury in Swine.

BACKGROUND: We evaluated the effects of two interventions that modify the red cell storage lesion on kidney and lung injury in experimental models of transfusion. METHODS: White-landrace pigs (n = 32) were allocated to receive sham transfusion (crystalloid), 14-day stored allogeneic red cells, 14-day red cells washed using the red cells washing/salvage system (CATS; Fresenius, Germany), or 14-day red cells rejuvenated using the inosine solution (Rejuvesol solution; Zimmer Biomet, USA) and washed using the CATS device. Functional, biochemical, and histologic markers of organ injury were assessed for up to 24 h posttransfusion. RESULTS: Transfusion of 14 day red cells resulted in lung injury (lung injury score vs. sham, mean difference -0.3 (95% CI, -0.6 to -0.1; P = 0.02), pulmonary endothelial dysfunction, and tissue leukocyte sequestration. Mechanical washing reduced red cell-derived microvesicles but increased cell-free hemoglobin in 14-day red cell units. Transfusion of washed red cells reduced leukocyte sequestration but did not reduce the lung injury score (mean difference -0.2; 95% CI, -0.5 to 0.1; P = 0.19) relative to 14-day cells. Transfusion of washed red cells also increased endothelial activation and kidney injury. Rejuvenation restored adenosine triphosphate to that of fresh red cells and reduced microvesicle concentrations without increasing cell-free hemoglobin release. Transfusion of rejuvenated red cells reduced plasma cell-free hemoglobin, leukocyte sequestration, and endothelial dysfunction in recipients and reduced lung and kidney injury relative to 14-day or washed 14-day cells. CONCLUSIONS: Reversal of the red cell storage lesion by rejuvenation reduces transfusion-associated organ injury in swine.

Comparison of the release of microRNAs and extracellular vesicles from platelets in response to different agonists.

On activation platelets release microRNAs and extracellular vesicles (EV) into circulation. The release of EV from platelets has been shown to be dependent on the agonist; in this study, we investigated whether the microRNA profile or EV released from platelets was also agonist specific. Washed platelets from healthy subjects were maximally stimulated with agonists specific for the receptors for collagen (Glycoprotein VI (GPVI)), thrombin (PAR1/PAR4), or ADP (P2Y1/P2Y12) with/without inhibiting secondary mediators, using aspirin to block cyclooxygenase-1 and apyrase to remove ADP. The released microRNAs were profiled using TaqMan microRNA microarray cards. Platelet-derived EV (pdEV) were characterized by size (Nanoparticle Tracking Analysis, NTA), for procoagulant activity (Annexin-V binding and support of thrombin generation), and for the EV markers CD63 and HSP70. Platelet activation triggered the release of 57-79 different microRNAs, dependent upon agonist, with a core of 46 microRNAs observed with all agonists. There was a high level of correlation between agonists (r2 > 0.98; p < 0.0001 for all), and with the microRNA content of the parent platelets (r2 > 0.98; p < 0.0001). The 46 microRNAs seen in all samples are predicted to have significant effects on the translation of proteins involved in endocytosis, cell cycle control, and differentiation. MiR-223-3p was the most abundant in all samples and has previously been implicated in myeloid lineage development and demonstrated to have anti-inflammatory effects. Stimulation through GPVI produced a pdEV population with significantly more procoagulant activity than the other agonists. Apyrase significantly reduced microRNA and pdEV release, while aspirin had little effect. These data suggest that all tested agonists trigger the release of a similar microRNA profile while the procoagulant activity of the pdEV was agonist dependent. ADP was shown to play an important role in the release of both microRNAs and pdEV.

New gene functions in megakaryopoiesis and platelet formation.

Platelets are the second most abundant cell type in blood and are essential for maintaining haemostasis. Their count and volume are tightly controlled within narrow physiological ranges, but there is only limited understanding of the molecular processes controlling both traits. Here we carried out a high-powered meta-analysis of genome-wide association studies (GWAS) in up to 66,867 individuals of European ancestry, followed by extensive biological and functional assessment. We identified 68 genomic loci reliably associated with platelet count and volume mapping to established and putative novel regulators of megakaryopoiesis and platelet formation. These genes show megakaryocyte-specific gene expression patterns and extensive network connectivity. Using gene silencing in Danio rerio and Drosophila melanogaster, we identified 11 of the genes as novel regulators of blood cell formation. Taken together, our findings advance understanding of novel gene functions controlling fate-determining events during megakaryopoiesis and platelet formation, providing a new example of successful translation of GWAS to function.

Histopathological evaluation of thrombus in patients presenting with stent thrombosis. A multicenter European study: a report of the prevention of late stent thrombosis by an interdisciplinary global European effort consortium.

BACKGROUND: Stent thrombosis (ST) is a rare but serious complication following percutaneous coronary intervention. Analysis of thrombus composition from patients undergoing catheter thrombectomy may provide important insights into the pathological processes leading to thrombus formation. We performed a large-scale multicentre study to evaluate thrombus specimens in patients with ST across Europe. METHODS: Patients presenting with ST and undergoing thrombus aspiration were eligible for inclusion. Thrombus collection was performed according to a standardized protocol and specimens were analysed histologically at a core laboratory. Serial tissue cross sections were stained with haematoxylin-eosin (H&E), Carstairs and Luna. Immunohistochemistry was performed to identify leukocyte subsets, prothrombotic neutrophil extracellular traps (NETs), erythrocytes, platelets, and fibrinogen. RESULTS: Overall 253 thrombus specimens were analysed; 79 (31.2%) from patients presenting with early ST, 174 (68.8%) from late ST; 79 (31.2%) were from bare metal stents, 166 (65.6%) from drug-eluting stents, 8 (3.2%) were from stents of unknown type. Thrombus specimens displayed heterogeneous morphology with platelet-rich thrombus and fibrin/fibrinogen fragments most abundant; mean platelet coverage was 57% of thrombus area. Leukocyte infiltrations were hallmarks of both early and late ST (early: 2260 ± 1550 per mm(2) vs. late: 2485 ± 1778 per mm(2); P = 0.44); neutrophils represented the most prominent subset (early: 1364 ± 923 per mm(2) vs. late: 1428 ± 1023 per mm(2); P = 0.81). Leukocyte counts were significantly higher compared with a control group of patients with thrombus aspiration in spontaneous myocardial infarction. Neutrophil extracellular traps were observed in 23% of samples. Eosinophils were present in all stent types, with higher numbers in patients with late ST in sirolimus-and everolimus-eluting stents. CONCLUSION: In a large-scale study of histological thrombus analysis from patients presenting with ST, thrombus specimens displayed heterogeneous morphology. Recruitment of leukocytes, particularly neutrophils, appears to be a hallmark of ST. The presence of NETs supports their pathophysiological relevance. Eosinophil recruitment suggests an allergic component to the process of ST.

A meta-analysis of genome-wide association studies identifies ORM1 as a novel gene controlling thrombin generation potential.

Thrombin, the major enzyme of the hemostatic system, is involved in biological processes associated with several human diseases. The capacity of a given individual to generate thrombin, called the thrombin generation potential (TGP), can be robustly measured in plasma and was shown to associate with thrombotic disorders. To investigate the genetic architecture underlying the interindividual TGP variability, we conducted a genome-wide association study in 2 discovery samples (N = 1967) phenotyped for 3 TGP biomarkers, the endogenous thrombin potential, the peak height, and the lag time, and replicated the main findings in 2 independent studies (N = 1254). We identified the ORM1 gene, coding for orosomucoid, as a novel locus associated with lag time variability, reflecting the initiation process of thrombin generation with a combined P value of P = 7.1 × 10(-15) for the lead single nucleotide polymorphism (SNP) (rs150611042). This SNP was also observed to associate with ORM1 expression in monocytes (P = 8.7 × 10(-10)) and macrophages (P = 3.2 × 10(-3)). In vitro functional experiments further demonstrated that supplementing normal plasma with increasing orosomucoid concentrations was associated with impaired thrombin generation. These results pave the way for novel mechanistic pathways and therapeutic perspectives in the etiology of thrombin-related disorders.

A trans-acting locus regulates an anti-viral expression network and type 1 diabetes risk.

Combined analyses of gene networks and DNA sequence variation can provide new insights into the aetiology of common diseases that may not be apparent from genome-wide association studies alone. Recent advances in rat genomics are facilitating systems-genetics approaches. Here we report the use of integrated genome-wide approaches across seven rat tissues to identify gene networks and the loci underlying their regulation. We defined an interferon regulatory factor 7 (IRF7)-driven inflammatory network (IDIN) enriched for viral response genes, which represents a molecular biomarker for macrophages and which was regulated in multiple tissues by a locus on rat chromosome 15q25. We show that Epstein-Barr virus induced gene 2 (Ebi2, also known as Gpr183), which lies at this locus and controls B lymphocyte migration, is expressed in macrophages and regulates the IDIN. The human orthologous locus on chromosome 13q32 controlled the human equivalent of the IDIN, which was conserved in monocytes. IDIN genes were more likely to associate with susceptibility to type 1 diabetes (T1D)-a macrophage-associated autoimmune disease-than randomly selected immune response genes (P = 8.85 × 10(-6)). The human locus controlling the IDIN was associated with the risk of T1D at single nucleotide polymorphism rs9585056 (P = 7.0 × 10(-10); odds ratio, 1.15), which was one of five single nucleotide polymorphisms in this region associated with EBI2 (GPR183) expression. These data implicate IRF7 network genes and their regulatory locus in the pathogenesis of T1D.

Genome-wide haplotype analysis of cis expression quantitative trait loci in monocytes.

In order to assess whether gene expression variability could be influenced by several SNPs acting in cis, either through additive or more complex haplotype effects, a systematic genome-wide search for cis haplotype expression quantitative trait loci (eQTL) was conducted in a sample of 758 individuals, part of the Cardiogenics Transcriptomic Study, for which genome-wide monocyte expression and GWAS data were available. 19,805 RNA probes were assessed for cis haplotypic regulation through investigation of ~2,1 × 10(9) haplotypic combinations. 2,650 probes demonstrated haplotypic p-values >10(4)-fold smaller than the best single SNP p-value. Replication of significant haplotype effects were tested for 412 probes for which SNPs (or proxies) that defined the detected haplotypes were available in the Gutenberg Health Study composed of 1,374 individuals. At the Bonferroni correction level of 1.2 × 10(-4) (~0.05/412), 193 haplotypic signals replicated. 1000 G imputation was then conducted, and 105 haplotypic signals still remained more informative than imputed SNPs. In-depth analysis of these 105 cis eQTL revealed that at 76 loci genetic associations were compatible with additive effects of several SNPs, while for the 29 remaining regions data could be compatible with a more complex haplotypic pattern. As 24 of the 105 cis eQTL have previously been reported to be disease-associated loci, this work highlights the need for conducting haplotype-based and 1000 G imputed cis eQTL analysis before commencing functional studies at disease-associated loci.

Hyperphosphatemia, Phosphoprotein Phosphatases, and Microparticle Release in Vascular Endothelial Cells.

Hyperphosphatemia in patients with advanced CKD is thought to be an important contributor to cardiovascular risk, in part because of endothelial cell (EC) dysfunction induced by inorganic phosphate (Pi). Such patients also have an elevated circulating concentration of procoagulant endothelial microparticles (MPs), leading to a prothrombotic state, which may contribute to acute occlusive events. We hypothesized that hyperphosphatemia leads to MP formation from ECs through an elevation of intracellular Pi concentration, which directly inhibits phosphoprotein phosphatases, triggering a global increase in phosphorylation and cytoskeletal changes. In cultured human ECs (EAhy926), incubation with elevated extracellular Pi (2.5 mM) led to a rise in intracellular Pi concentration within 90 minutes. This was mediated by PiT1/slc20a1 Pi transporters and led to global accumulation of tyrosine- and serine/threonine-phosphorylated proteins, a marked increase in cellular Tropomyosin-3, plasma membrane blebbing, and release of 0.1- to 1-µm-diameter MPs. The effect of Pi was independent of oxidative stress or apoptosis. Similarly, global inhibition of phosphoprotein phosphatases with orthovanadate or fluoride yielded a global protein phosphorylation response and rapid release of MPs. The Pi-induced MPs expressed VE-cadherin and superficial phosphatidylserine, and in a thrombin generation assay, they displayed significantly more procoagulant activity than particles derived from cells incubated in medium with a physiologic level of Pi (1 mM). These data show a mechanism of Pi-induced cellular stress and signaling, which may be widely applicable in mammalian cells, and in ECs, it provides a novel pathologic link between hyperphosphatemia, generation of MPs, and thrombotic risk.

DNA methylation and body-mass index: a genome-wide analysis.

BACKGROUND: Obesity is a major health problem that is determined by interactions between lifestyle and environmental and genetic factors. Although associations between several genetic variants and body-mass index (BMI) have been identified, little is known about epigenetic changes related to BMI. We undertook a genome-wide analysis of methylation at CpG sites in relation to BMI. METHODS: 479 individuals of European origin recruited by the Cardiogenics Consortium formed our discovery cohort. We typed their whole-blood DNA with the Infinium HumanMethylation450 array. After quality control, methylation levels were tested for association with BMI. Methylation sites showing an association with BMI at a false discovery rate q value of 0·05 or less were taken forward for replication in a cohort of 339 unrelated white patients of northern European origin from the MARTHA cohort. Sites that remained significant in this primary replication cohort were tested in a second replication cohort of 1789 white patients of European origin from the KORA cohort. We examined whether methylation levels at identified sites also showed an association with BMI in DNA from adipose tissue (n=635) and skin (n=395) obtained from white female individuals participating in the MuTHER study. Finally, we examined the association of methylation at BMI-associated sites with genetic variants and with gene expression. FINDINGS: 20 individuals from the discovery cohort were excluded from analyses after quality-control checks, leaving 459 participants. After adjustment for covariates, we identified an association (q value ≤0·05) between methylation at five probes across three different genes and BMI. The associations with three of these probes--cg22891070, cg27146050, and cg16672562, all of which are in intron 1 of HIF3A--were confirmed in both the primary and second replication cohorts. For every 0·1 increase in methylation ß value at cg22891070, BMI was 3·6% (95% CI 2·4-4·9) higher in the discovery cohort, 2·7% (1·2-4·2) higher in the primary replication cohort, and 0·8% (0·2-1·4) higher in the second replication cohort. For the MuTHER cohort, methylation at cg22891070 was associated with BMI in adipose tissue (p=1·72 × 10(-5)) but not in skin (p=0·882). We observed a significant inverse correlation (p=0·005) between methylation at cg22891070 and expression of one HIF3A gene-expression probe in adipose tissue. Two single nucleotide polymorphisms--rs8102595 and rs3826795--had independent associations with methylation at cg22891070 in all cohorts. However, these single nucleotide polymorphisms were not significantly associated with BMI. INTERPRETATION: Increased BMI in adults of European origin is associated with increased methylation at the HIF3A locus in blood cells and in adipose tissue. Our findings suggest that perturbation of hypoxia inducible transcription factor pathways could have an important role in the response to increased weight in people. FUNDING: The European Commission, National Institute for Health Research, British Heart Foundation, and Wellcome Trust.

Identification of secreted phosphoprotein 1 gene as a new rheumatoid arthritis susceptibility gene.

OBJECTIVE: To evaluate the contribution of the SPP1 rs11439060 and rs9138 polymorphisms, previously reported as autoimmune risk variants, in the rheumatoid arthritis (RA) genetic background according to anti-citrullinated protein antibodies (ACPAs) status of RA individuals. METHODS: We analysed a total of 11,715 RA cases and 26,493 controls from nine independent cohorts; all individuals were genotyped or had imputed genotypes for SPP1 rs11439060 and rs9138. The effect of the SPP1 rs11439060 and rs9138 risk-allele combination on osteopontin (OPN) expression in macrophages and OPN serum levels was investigated. RESULTS: We provide evidence for a distinct contribution of SPP1 to RA susceptibility according to ACPA status: the combination of ≥3 SPP1 rs11439060 and rs9138 common alleles was associated mainly with ACPA negativity (p=1.29×10(-5), ORACPA-negative 1.257 (1.135 to 1.394)) and less with ACPA positivity (p=0.0148, ORACPA-positive 1.072 (1.014 to 1.134)). The ORs between these subgroups (ie, ACPA-positive and ACPA-negative) significantly differed (p=7.33×10(-3)). Expression quantitative trait locus analysis revealed an association of the SPP1 risk-allele combination with decreased SPP1 expression in peripheral macrophages from 599 individuals. To corroborate these findings, we found an association of the SPP1 risk-allele combination and low serum level of secreted OPN (p=0.0157), as well as serum level of secreted OPN correlated positively with ACPA production (p=0.005; r=0.483). CONCLUSIONS: We demonstrate a significant contribution of the combination of SPP1 rs11439060 and rs9138 frequent alleles to risk of RA, the magnitude of the association being greater in patients negative for ACPAs.

Allelic expression mapping across cellular lineages to establish impact of non-coding SNPs.

Most complex disease-associated genetic variants are located in non-coding regions and are therefore thought to be regulatory in nature. Association mapping of differential allelic expression (AE) is a powerful method to identify SNPs with direct cis-regulatory impact (cis-rSNPs). We used AE mapping to identify cis-rSNPs regulating gene expression in 55 and 63 HapMap lymphoblastoid cell lines from a Caucasian and an African population, respectively, 70 fibroblast cell lines, and 188 purified monocyte samples and found 40-60% of these cis-rSNPs to be shared across cell types. We uncover a new class of cis-rSNPs, which disrupt footprint-derived de novo motifs that are predominantly bound by repressive factors and are implicated in disease susceptibility through overlaps with GWAS SNPs. Finally, we provide the proof-of-principle for a new approach for genome-wide functional validation of transcription factor-SNP interactions. By perturbing NFκB action in lymphoblasts, we identified 489 cis-regulated transcripts with altered AE after NFκB perturbation. Altogether, we perform a comprehensive analysis of cis-variation in four cell populations and provide new tools for the identification of functional variants associated to complex diseases.

Variation in thromboxane B2 concentrations in serum and plasma in patients taking regular aspirin before and after clopidogrel therapy.

Dual antiplatelet therapy with aspirin and a P2Y12 antagonist is widely prescribed for the prevention of thrombotic events in patients with an acute coronary syndrome or undergoing percutaneous coronary intervention (PCI). It is recognised that there is inter-individual variation in the antiplatelet effects of both drugs. Recent data also suggest that P2Y12 antagonists can affect the response to aspirin. A direct indicator of the effect of aspirin on platelets is their ability to generate thromboxane, which if measured as the difference between the level of thromboxane B2 in serum and plasma ([TxB2]S-P) avoids the confounding effect of endogenous TxB2 production from other cells. We therefore analysed [TxB2]S-P as a measure of aspirin response in a group of 123 patients undergoing elective PCI before and after the introduction of clopidogrel. In a subgroup of 40 patients taking aspirin alone, we compared [TxB2]S-P and VerifyNow Aspirin for the assessment of aspirin response. There was a wide variation in plasma and serum TxB2 concentrations both before and after clopidogrel therapy but only 3.5% of patients had residual serum concentration of TxB2 > 10 ng/ml. There was a strong correlation between the pre and post clopidogrel levels of TxB2 (r ≥ 0.78; p = 0.001) and no significant difference in [TxB2]S-P. There was no correlation between the magnitude of response to clopidogrel response and the generation of thromboxane B2. Correlation between [TxB2]S-P and VerifyNow Aspirin was poor. We conclude that the use of a P2Y12 antagonist does not influence the effect of aspirin on the ability of platelets to generate thromboxane. Therefore, measurement of TxB2 levels in serum, after subtracting the contribution from plasma, provides a measure of the response to aspirin in patients taking dual antiplatelet therapy.

Long-range DNA looping and gene expression analyses identify DEXI as an autoimmune disease candidate gene.

The chromosome 16p13 region has been associated with several autoimmune diseases, including type 1 diabetes (T1D) and multiple sclerosis (MS). CLEC16A has been reported as the most likely candidate gene in the region, since it contains the most disease-associated single-nucleotide polymorphisms (SNPs), as well as an imunoreceptor tyrosine-based activation motif. However, here we report that intron 19 of CLEC16A, containing the most autoimmune disease-associated SNPs, appears to behave as a regulatory sequence, affecting the expression of a neighbouring gene, DEXI. The CLEC16A alleles that are protective from T1D and MS are associated with increased expression of DEXI, and no other genes in the region, in two independent monocyte gene expression data sets. Critically, using chromosome conformation capture (3C), we identified physical proximity between the DEXI promoter region and intron 19 of CLEC16A, separated by a loop of >150 kb. In reciprocal experiments, a 20 kb fragment of intron 19 of CLEC16A, containing SNPs associated with T1D and MS, as well as with DEXI expression, interacted with the promotor region of DEXI but not with candidate DNA fragments containing other potential causal genes in the region, including CLEC16A. Intron 19 of CLEC16A is highly enriched for transcription-factor-binding events and markers associated with enhancer activity. Taken together, these data indicate that although the causal variants in the 16p13 region lie within CLEC16A, DEXI is an unappreciated autoimmune disease candidate gene, and illustrate the power of the 3C approach in progressing from genome-wide association studies results to candidate causal genes.