Search

Identification of disordered platelet function is important to guide peri-operative bleeding management as well as long term treatment and prognostic strategies in individuals with platelet bleeding disorders. Light transmission aggregometry (LTA), the current gold standard diagnostic test of platelet function is a time consuming technique almost exclusively performed in specialised laboratories and almost universally unavailable in regional centres in Australia, where there is an unmet need for access to specialised platelet function diagnostic services. 96-well plate-based aggregometry (Optimul, UK), has been utilised in research laboratories as a novel platform to investigate platelet function. We evaluated the Optimul assay at two centres in Australia, one regional and one tertiary metropolitan, to assess its feasibility as a screening test applicable to remote regional centres. Concentration-response curves were established from 45 healthy volunteers at the participating regional hospital and from 31 healthy volunteers at the tertiary institution. Optimul successfully detected anti-platelet effects in individuals taking aspirin (n=4), NSAID (n=2), clopidogrel (n=2) and dual therapy with aspirin and clopidogrel (n=1). When tested in parallel to LTA in individuals referred for the evaluation of abnormal bleeding symptoms there was overall a very good level of agreement between Optimul and LTA [Cohen's kappa (k

Background Platelets circulate in the blood of healthy individuals for approximately 7-10 days regulated by finely balanced processes of production and destruction. As platelets are anucleate we reasoned that their protein composition would change as they age and that this change would be linked to alterations in structure and function. Objective To isolate platelets of different ages from healthy individuals to test the hypothesis that changes in protein content cause alterations in platelet structure and function. Methods Platelets were separated according to thiazole orange fluorescence intensity as a surrogate indicator of mRNA content and so a marker of platelet age and then subjected to proteomics, imaging, and functional assays to produce an in-depth analysis of platelet composition and function. Results Total protein content was 45±5% lower in old platelets compared to young platelets. Predictive proteomic pathway analysis identified associations with 28 biological processes, notably higher hemostasis in young platelets whilst apoptosis and senescence were higher in old platelets. Further studies confirmed platelet ageing was linked to a decrease in cytoskeletal protein and associated capability to spread and adhere, a reduction in mitochondria number, and lower calcium dynamics and granule secretion. Conclusions Our findings demonstrate changes in protein content are linked to alterations in function as platelets age. This work delineates physical and functional changes in platelets as they age and serves as a base to examine differences associated with altered mean age of platelet populations in conditions such as immune thrombocytopenia and diabetes.

Background Antiplatelet therapy (APT) has improved cardiovascular outcomes, but some patients develop thrombosis despite APT. Adjusting APT to platelet reactivity with conventional platelet reactivity tests has proven unsuccessful. Many circulating noncoding RNAs (ncRNAs) are derived from platelets and could serve as novel platelet function markers. Material and methods Platelet reactivity was assessed using light transmission aggregometry (LTA) in the Bruneck 2015 study (N = 338), using the agonists arachidonic acid, adenosine diphosphate, collagen, TRAP-6 amide and U46619. LTA platelet releasates were then used for RT-qPCR of five platelet-enriched microRNAs, three circular RNAs and two long non-coding RNAs. Platelet-poor plasma (PPP) was used as negative control. Results and conclusions Platelet agonists induced aggregation and ncRNA release, with aspirin takers (N = 155) showing lower ncRNA release than individuals not on aspirin (N = 183). Agonist responsiveness differed among ncRNAs, with miR-150 being hyperresponsive to adenosine diphosphate and miR-21 being hyperresponsive to arachidonic acid, whereas other ncRNAs were most strongly released to collagen, suggesting a selective release mechanism. In individuals not on aspirin, the inflammation markers C-reactive protein and granulocyte counts correlated positively with platelet-derived ncRNAs in PPP, while they correlated inversely with platelet-derived ncRNAs in releasates. These correlations were not present in aspirin takers. Higher PPP levels and lower releasate levels of platelet-derived ncRNAs in inflammation suggest platelet exhaustion ex vivo due to platelet pre-activation in vivo. Funding sources British Heart Foundation, VASCage (Centre for Promoting Vascular Health in the Ageing Community) of the Austrian Research Promotion Agency FFG (COMET program - Competence Centers for Excellent Technologies).

Background and purpose P2Y12 receptor antagonists reduce platelet aggregation and the incidence of arterial thrombosis. Adenosine signalling in platelets directly affects cyclic nucleotide tone, which we have shown to have a synergistic relationship with P2Y12 inhibition. Several studies suggest that ticagrelor inhibits erythrocyte uptake of adenosine and that this could also contribute to its antiplatelet effects. We therefore examined the effects on platelet activation of adenosine signalling activators in combination with the P2Y12 receptor antagonists ticagrelor and prasugrel. Experimental approach Human washed platelets, platelet-rich plasma and whole blood were used to test the interactions between ticagrelor or prasugrel and adenosine or 5'-N-ethylcarboxamidoadenosine (NECA). Platelet reactivity to thrombin, protease-activated receptor 1 (PAR-1) activation or collagen was assessed by a combination of 96-well plate aggregometry, light transmission aggregometry, whole blood aggregometry, ATP release assay and levels of cAMP. Key results The inhibitory effects of ticagrelor and prasugrel on platelet aggregation and ATP release were enhanced in the presence of adenosine or NECA. Isobolographic analysis indicated a powerful synergy between P2Y12 receptor inhibition and adenosine signalling activators. Increased levels of cAMP in platelets were also observed. In all cases, ticagrelor showed similar synergistic effects on platelet inhibition as prasugrel in the presence of adenosine or NECA. Conclusion and implications These results indicate that P2Y12 antagonists have a synergistic relationship with adenosine signalling and that their efficacy may depend partly upon the presence of endogenous adenosine. This effect was common for prasugrel and ticagrelor despite reports of differences in their effects upon adenosine reuptake.

This 2020 International Consensus on Cardiopulmonary Resuscitation and Emergency Cardiovascular Care Science With Treatment Recommendations (CoSTR) for pediatric life support is based on the most extensive evidence evaluation ever performed by the Pediatric Life Support Task Force. Three types of evidence evaluation were used in this review: systematic reviews, scoping reviews, and evidence updates. Per agreement with the evidence evaluation recommendations of the International Liaison Committee on Resuscitation, only systematic reviews could result in a new or revised treatment recommendation. Systematic reviews performed for this 2020 CoSTR for pediatric life support included the topics of sequencing of airway-breaths-compressions versus compressions-airway-breaths in the delivery of pediatric basic life support, the initial timing and dose intervals for epinephrine administration during resuscitation, and the targets for oxygen and carbon dioxide levels in pediatric patients after return of spontaneous circulation. The most controversial topics included the initial timing and dose intervals of epinephrine administration (new treatment recommendations were made) and the administration of fluid for infants and children with septic shock (this latter topic was evaluated by evidence update). All evidence reviews identified the paucity of pediatric data and the need for more research involving resuscitation of infants and children.

We have identified a rare missense variant on chromosome 9, position 125145990 (GRCh37), in exon 8 in PTGS1 (the gene encoding cyclo-oxygenase 1, COX-1, the target of anti-thrombotic aspirin therapy). We report that in the homozygous state within a large consanguineous family this variant is associated with a bleeding phenotype and alterations in platelet reactivity and eicosanoid production. Western blotting and confocal imaging demonstrated that COX-1 was absent in the platelets of three family members homozygous for the PTGS1 variant but present in their leukocytes. Platelet reactivity, as assessed by aggregometry, lumi-aggregometry and flow cytometry, was impaired in homozygous family members, as were platelet adhesion and spreading. The productions of COX-derived eicosanoids by stimulated platelets were greatly reduced but there were no changes in the levels of urinary metabolites of COX-derived eicosanoids. The proband exhibited additional defects in platelet aggregation and spreading which may explain why her bleeding phenotype was slightly more severe than those of other homozygous affected relatives. This is the first demonstration in humans of the specific loss of platelet COX-1 activity and provides insight into its consequences for platelet function and eicosanoid metabolism. Notably despite the absence of thromboxane A2 (TXA2) formation by platelets, urinary TXA2 metabolites were in the normal range indicating these cannot be assumed as markers of in vivo platelet function. Results from this study are important benchmarks for the effects of aspirin upon platelet COX-1, platelet function and eicosanoid production as they define selective platelet COX-1 ablation within humans.

Background Hypoxia resulting from ascent to high-altitude or pathological states at sea level is known to increase platelet reactivity. Previous work from our group has suggested that this may be adenosine diphosphate (ADP)-specific. Given the clinical importance of drugs targeting ADP pathways, research into the impact of hypoxia on platelet ADP pathways is highly important. Methods Optimul aggregometry was performed on plasma from 29 lowland residents ascending to 4,700 m, allowing systematic assessment of platelet reactivity in response to several platelet agonists. Aggregometry was also performed in response to ADP in the presence of inhibitors of the two main ADP receptors, P2Y1 and P2Y12 (MRS2500 and cangrelor, respectively). Phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a key determinant of platelet aggregation, was analysed using the VASPFix assay. Results Hypobaric hypoxia significantly reduced the ability of a fixed concentration of cangrelor to inhibit ADP-induced aggregation and increased basal VASP phosphorylation. However, in the absence of P2Y receptor inhibitors, we did not find evidence of increased platelet sensitivity to any of the agonists tested and found reduced sensitivity to thrombin receptor-activating peptide-6 amide. Conclusion Our results provide evidence of increased P2Y1 receptor activity at high altitude and suggest down-regulation of the P2Y12 pathway through increased VASP phosphorylation. These changes in ADP pathway activity are of potential therapeutic significance to high-altitude sojourners and hypoxic sea level patients prescribed platelet inhibitors and warrant further investigation.

Objective: Platelets are central to acute myocardial infarction (MI). How the platelet proteome is altered during MI is unknown. We sought to describe changes in the platelet proteome during MI and identify corresponding functional consequences. Approach and Results: Platelets from patients experiencing ST-segment–elevation MI (STEMI) before and 3 days after treatment (n=30) and matched patients with severe stable coronary artery disease before and 3 days after coronary artery bypass grafting (n=25) underwent quantitative proteomic analysis. Elevations in the proteins S100A8 and S100A9 were detected at the time of STEMI compared with stable coronary artery disease (S100A8: FC, 2.00; false discovery rate, 0.05; S100A9: FC, 2.28; false discovery rate, 0.005). During STEMI, only S100A8 mRNA and protein levels were correlated in platelets ( R =0.46, P =0.012). To determine whether de novo protein synthesis occurs, activated platelets were incubated with 13C-labeled amino acids for 24 hours and analyzed by mass spectrometry. No incorporation was confidently detected. Platelet S100A8 and S100A9 was strongly correlated with neutrophil abundance at the time of STEMI. When isolated platelets and neutrophils were coincubated under quiescent and activated conditions, release of S100A8 from neutrophils resulted in uptake of S100A8 by platelets. Neutrophils released S100A8/A9 as free heterodimer, rather than in vesicles or extracellular traps. In the community-based Bruneck study (n=338), plasma S100A8/A9 was inversely associated with platelet reactivity—an effect abrogated by aspirin. Conclusions: Leukocyte-to-platelet protein transfer may occur in a thromboinflammatory environment such as STEMI. Plasma S100A8/A9 was negatively associated with platelet reactivity. These findings highlight neutrophils as potential modifiers for thrombotic therapies in coronary artery disease.

Host genetic variants influence the susceptibility and severity of several infectious diseases, and the discovery of genetic associations with coronavirus disease 2019 (COVID-19) phenotypes could help to develop new therapeutic strategies to decrease its burden. Between May 2020 and June 2021, we used COVID-19 data released periodically by UK Biobank and performed 65 genome-wide association studies in up to 18 releases of COVID-19 susceptibility (n = 18,481 cases in June 2021), hospitalization (n = 3,260), severe outcomes (n = 1,244), and deaths (n = 1,104), stratified by sex and ancestry. In coherence with previous studies, we observed two independent signals at the chr3p21.31 locus (rs73062389-A, odds ratio [OR], 1.21 (P = 4.26 × 10

P2YHuman washed platelets, platelet-rich plasma and whole blood were used to test the interactions between ticagrelor or prasugrel and adenosine or 5'-N-ethylcarboxamidoadenosine (NECA). Platelet reactivity to thrombin, protease-activated receptor 1 (PAR-1) activation or collagen was assessed by a combination of 96-well plate aggregometry, light transmission aggregometry, whole blood aggregometry, ATP release assay and levels of cAMP.The inhibitory effects of ticagrelor and prasugrel on platelet aggregation and ATP release were enhanced in the presence of adenosine or NECA. Isobolographic analysis indicated a powerful synergy between P2YThese results indicate that P2Y

Host genetic variants influence the susceptibility and severity of several infectious diseases, and the discovery of novel genetic associations with Covid-19 phenotypes could help developing new therapeutic strategies to reduce its burden.Between May 2020 and June 2021, we used Covid-19 data released periodically by UK Biobank and performed 65 Genome-Wide Association Studies (GWAS) in up to 18 releases of Covid-19 susceptibility (N=18,481 cases in June 2021), hospitalization (N=3,260), severe outcomes (N=1,244) and death (N=1,104), stratified by sex and ancestry.In coherence with previous studies, we observed 2 independent signals at the chr3p21.31 locus (rs73062389-A, OR=1.21, P=4.26×10−15 and rs71325088-C, OR=1.62, P=2.25×10−9) modulating susceptibility and severity, respectively, and a signal influencing susceptibility at the ABO locus (rs9411378-A, OR=1.10, P=3.30×10−12), suggesting an increased risk of infection in non-O blood groups carriers. Additional signals at the APOE (associated with severity and death) LRMDA (susceptibility in non-European) and chr2q32.3 (susceptibility in women) loci were also identified but did not replicate in independent datasets. We then devised an original approach to extract variants exhibiting an increase in significance over time. When applied to the susceptibility, hospitalization and severity analyses, this approach revealed the known DPP9, RPL24 and MAPT loci, amongst thousands of other signals. Finally, this significance trajectory analysis was applied to the larger Covid-19hgi meta-analyses, where additional loci of interest, related to the immune system, were identified.These results, freely available on the GRASP portal, provide new insights on the genetic mechanisms involved in Covid-19 phenotypes.

During platelet activation, arachidonic acid (AA) is released from membrane phospholipids and metabolized to thromboxane A2 (TXA2) through the actions of cyclooxygenase-1 (COX-1) and TXA2 synthase. Note, TXA2 binds to the platelet TXA2 receptor, causing shape change, secretion and platelet aggregation.1 Also, COX-1 (599aa; 70 kDa) has cyclooxygenase and peroxidase activities and it is functionally active as a homodimer, with each COX-1 monomer consisting of four highly conserved domains: an N-terminal signal peptide, a dimerization domain, a membrane-binding domain (MBD) and a large C-terminal catalytic domain2 (Figure 1A). Irreversible COX-1 inhibition by aspirin is a widely established anti-platelet therapy in cardiovascular disease., Fundación Mutua Madrileña, Grant/Award Number: AP172142019; Fundación Séneca, Grant/Award Number: 19873/GERM/15; Gerencia Regional de Salud, Grant/Award Numbers: 1647/A/17, 2061A/19; Instituto de Salud Carlos III (ISCIII) & Feder, Grant/Award Numbers: CB15/00055, PI17/01966, PI18/00598, PI20/00926, PI17/01311; Junta de Castilla y León; British Heart Foundation, Grant/Award Number: PG/17/40/33028; Ayuda a Grupos de Trabajo en Patología Hemorrágica; Premio López Borrasca 2019; Sociedad Española de Trombosis y Hemostasia.

Background Endothelium-derived prostacyclin and nitric oxide elevate platelet cyclic nucleotide levels and maintain quiescence. We previously demonstrated that a synergistic relationship exists between cyclic nucleotides and P2Y12 receptor inhibition. A number of clinically approved drug classes can modulate cyclic nucleotide tone in platelets including activators of NO-sensitive guanylyl cyclase (GC) and phosphodiesterase (PDE) inhibitors. However, the doses required to inhibit platelets produce numerous side effects including headache. Objective We investigated using GC-activators in combination with P2Y12 receptor antagonists as a way to selectively amplify the anti-thrombotic effect of both drugs. Methods In vitro light transmission aggregation and platelet adhesion under flow were performed on washed platelets and platelet rich plasma. Aggregation in whole blood and a ferric chloride-induced arterial thrombosis model were also performed. Results The GC-activator BAY-70 potentiated the action of the P2Y12 receptor inhibitor prasugrel active metabolite in aggregation and adhesion studies and was associated with raised intra-platelet cyclic nucleotide levels. Furthermore, mice administered sub-maximal doses of the GC activator cinaciguat together with the PDE inhibitor dipyridamole and prasugrel, showed significant inhibition of ex vivo platelet aggregation and significantly reduced in vivo arterial thrombosis in response to injury without alteration in basal carotid artery blood flow. Conclusions Using in vitro, ex vivo, and in vivo functional studies, we show that low dose GC activators synergize with P2Y12 inhibition to produce powerful anti-platelet effects without altering blood flow. Therefore, modulation of intra-platelet cyclic nucleotide levels alongside P2Y12 inhibition can provide a strong, focused anti-thrombotic regimen while minimizing vasodilator side effects.

This 2019 focused update to the American Heart Association pediatric advanced life support guidelines follows the 2018 and 2019 systematic reviews performed by the Pediatric Life Support Task Force of the International Liaison Committee on Resuscitation. It aligns with the continuous evidence review process of the International Liaison Committee on Resuscitation, with updates published when the International Liaison Committee on Resuscitation completes a literature review based on new published evidence. This update provides the evidence review and treatment recommendations for advanced airway management in pediatric cardiac arrest, extracorporeal cardiopulmonary resuscitation in pediatric cardiac arrest, and pediatric targeted temperature management during post–cardiac arrest care. The writing group analyzed the systematic reviews and the original research published for each of these topics. For airway management, the writing group concluded that it is reasonable to continue bag-mask ventilation (versus attempting an advanced airway such as endotracheal intubation) in patients with out-of-hospital cardiac arrest. When extracorporeal membrane oxygenation protocols and teams are readily available, extracorporeal cardiopulmonary resuscitation should be considered for patients with cardiac diagnoses and in-hospital cardiac arrest. Finally, it is reasonable to use targeted temperature management of 32°C to 34°C followed by 36°C to 37.5°C, or to use targeted temperature management of 36°C to 37.5°C, for pediatric patients who remain comatose after resuscitation from out-of-hospital cardiac arrest or in-hospital cardiac arrest.

We have identified a rare missense variant on chromosome 9, position 125145990 (GRCh37), in exon 8 in prostaglandin endoperoxide synthase 1 (PTGS1) (the gene encoding cyclo-oxygenase 1 [COX-1], the target of anti-thrombotic aspirin therapy). We report that in the homozygous state within a large consanguineous family this variant is associated with a bleeding phenotype and alterations in platelet reactivity and eicosanoid production. Western blotting and confocal imaging demonstrated that COX-1 was absent in the platelets of three family members homozygous for the PTGS1 variant but present in their leukocytes. Platelet reactivity, as assessed by aggregometry, lumi-aggregometry and flow cytometry, was impaired in homozygous family members, as were platelet adhesion and spreading. The productions of COXderived eicosanoids by stimulated platelets were greatly reduced but there were no changes in the levels of urinary metabolites of COX-derived eicosanoids. The proband exhibited additional defects in platelet aggregation and spreading which may explain why her bleeding phenotype was slightly more severe than those of other homozygous affected relatives. This is the first demonstration in humans of the specific loss of platelet COX-1 activity and provides insight into its consequences for platelet function and eicosanoid metabolism. Notably despite the absence of thromboxane A2 formation by platelets, urinary thromboxane A2 metabolites were in the normal range indicating these cannot be assumed as markers of in vivo platelet function. Results from this study are important benchmarks for the effects of aspirin upon platelet COX-1, platelet function and eicosanoid production as they define selective platelet COX-1 ablation within humans.

Endothelium-derived prostacyclin and nitric oxide elevate platelet cyclic nucleotide levels and maintain quiescence. We previously demonstrated that a synergistic relationship exists between cyclic nucleotides and P2YWe investigated using GC-activators in combination with P2YIn vitro light transmission aggregation and platelet adhesion under flow were performed on washed platelets and platelet rich plasma. Aggregation in whole blood and a ferric chloride-induced arterial thrombosis model were also performed.The GC-activator BAY-70 potentiated the action of the P2YUsing in vitro, ex vivo, and in vivo functional studies, we show that low dose GC activators synergize with P2Y

Fibrosis is a major contributor to organ disease for which no specific therapy is available. MicroRNA-21 (miR-21) has been implicated in the fibrogenetic response, and inhibitors of miR-21 are currently undergoing clinical trials. Here, we explore how miR-21 inhibition may attenuate fibrosis using a proteomics approach. Transfection of miR-21 mimic or inhibitor in murine cardiac fibroblasts revealed limited effects on extracellular matrix (ECM) protein secretion. Similarly, miR-21–null mouse hearts showed an unaltered ECM composition. Thus, we searched for additional explanations as to how miR-21 might regulate fibrosis. In plasma samples from the community-based Bruneck Study, we found a marked correlation of miR-21 levels with several platelet-derived profibrotic factors, including TGF-β1. Pharmacological miR-21 inhibition with an antagomiR reduced the platelet release of TGF-β1 in mice. Mechanistically, Wiskott-Aldrich syndrome protein, a negative regulator of platelet TGF-β1 secretion, was identified as a direct target of miR-21. miR-21–null mice had lower platelet and leukocyte counts compared with littermate controls but higher megakaryocyte numbers in the bone marrow. Thus, to our knowledge this study reports a previously unrecognized effect of miR-21 inhibition on platelets. The effect of antagomiR-21 treatment on platelet TGF-β1 release, in particular, may contribute to the antifibrotic effects of miR-21 inhibitors., MicroRNA-21 inhibition may convey its therapeutic benefits in fibrosis through its action in bone marrow cells rather than targeting fibroblasts directly.

Introduction: Fibrosis is a major contributor to cardiac disease. MicroRNA-21 (miR-21) has been implicated as a regulator of fibrosis, with inhibitors of miR-21 currently undergoing clinical trials. We aimed to explore how miR-21 inhibition attenuates fibrosis using a proteomics approach. Methods and Results: As expected, transfection with miR-21 mimics and inhibitors altered the proliferation of murine cardiac fibroblasts. However, proteomic analysis of their conditioned media revealed limited effects on ECM secretion. Similarly, proteomic analysis of hearts from miR-21 null mice showed no major difference in extracellular matrix (ECM) composition. Thus, we searched for additional explanations as to how miR-21 might regulate fibrosis. In the community-based Bruneck study (year 2000 follow-up, n=671), we compared circulating miR-21 with a panel of 229 proteins associated with cardiovascular disease. Several platelet-derived pro-fibrotic factors correlated with plasma miR-21 levels, including the latency-associated peptide of transforming growth factor beta-1 (TGF-β1). This correlation was confirmed by ELISA measurements of active TGF-β1 in plasma (Bruneck 2015 follow-up, n=332). In agreement with this observation in men, systemic inhibition of miR-21 with an antagomiR reduced the platelet release of TGF-β1 in mice. Mechanistically, Wiskott-Aldrich Syndrome protein, a negative regulator of platelet TGF-β1 secretion, was identified as a direct target of miR-21. AntagomiR-21 treatment, however, had no effect on the platelet count or their aggregation response. In contrast, miR-21 null mice had significantly lower platelet counts compared to littermate controls. Conclusions: This study reports a previously unrecognized effect of miR-21 inhibition on platelets. Both genetic deletion of miR-21 and antagomiR treatment affect platelets. The effect of miR-21 inhibition on platelet TGF-β1 release, in particular, may contribute to the anti-fibrotic effects of miR-21 inhibitors.

Introduction: Thromboxane A2 [TxA2] is generated from arachidonic acid by cyclooxigenase-1 (COX-1) (prostaglandin H synthase-1) and thromboxane synthase. Aspirin, which irreversibly inhibits COX-1, is a widely used antiplatelet therapy with proven clinical efficacy. Inherited platelet disorders (IPD) are rare diseases caused by alterations of relevant genes in platelet formation and/or function. Despite the relevance of the TxA2 pathway in platelet physiology, few patients with mutations in PTGS1, the gene encoding COX-1, have been identified ( Objective: Characterization of a patient with aspirin-like platelet defect and moderate bleeding, enrolled in the Spanish multicentric project "Functional and molecular characterization of patients with IPD". Methods: The index case is a 13-year-old adopted girl of Asian origin, referred because of moderate chronic bleeding (BAT-ISTH=6) and an aspirin-like platelet dysfunction. No coagulation defect or other relevant clinical symptoms were present. Platelet phenotyping included: blood count, PFA-100; platelet aggregation [LTA], glycoproteins (GP), activation and secretion of granules by flow cytometry (FC), TxA2 synthesis by enzyme-immunoassay, synthesis of eicosanoids by tandem gas chromatography with mass spectrometry (LC-MS), western-blot (WB) of platelet lysates, and immunofluorescence (IF) assays. The patient's DNA was analyzed with a HTS-gene panel (Bastida et al, Haematologica 2018). A HEK 293T cell transfection model was established to further assess the pathogenicity of the candidate variant found in the patient. Results: The index case has normal platelet size and count (206x109/L; 11.4 fL). PFA-100 times were normal for COL-ADP and prolonged for COL-EPI (>300s). The FC analysis showed normal expression of GPs (Ib/IX, IIb/IIIa, Ia, GPVI) and reduced fibrinogen*488 binding (20-30%) in response to ADP, TRAP and low dose CRP (2ug/mL). P-selectin and CD63 secretion with agonists was comparable to those of controls. LTA was normal with ristocetin (1.25mg/mL) and TRAP (25uM), reduced by 40-50% with ADP (10uM) and collagen (3ug/mL) and absent with epinephrine (10uM), low dose collagen (1ug/mL) and arachidonic acid (1.6mM). LTA with U46619 (5uM), a direct agonist of the TxA2 receptor, was normal, suggesting a defect in TxA2 synthesis. Indeed, TxA2 levels in LTA supernatants in the patient were very low (5ng/mL; G, [p.Asn143Ser] in PTGS1. This variant, not previously described, affects a conserved residue in the catalytic domain of COX-1, which is one of the three N-glycosylation sites in the enzyme. The variant was not associated with reduced COX-1 expression as evaluated by WB in platelet lysates, and by IF in spread washed platelets and leukocytes. HEK 293T cells transfected with wild-type COX-1 construct (validated by RT-PCR and WB), displayed substantial TxA2 synthesis (500ng/mL; 2.5x105 transfected cells) in response to arachidonic acid. In contrast, similar transfection of p.143Ser COX-1 mutant almost abrogated this TxA2 production (≈50-75ng/mL in 2.5x105 transfected cells). Conclusion: We have identified a novel autosomal dominant COX-1 variant, p.Asn143Ser, associated with functional haploinsufficiency of the enzyme and platelet aggregation defects. To our knowledge, this case represents the third description of variants in PTGS1 (Nance, JTH 2016; Sivapalaratnam, Blood 2018), which cause platelet dysfunction and bleeding. Disclosures Almarza: Rocket Pharmaceuticals: Equity Ownership, Patents & Royalties, Research Funding. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Equity Ownership, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding.

Introduction MicroRNA-21 (miR-21) has been implicated as a regulator of fibrosis. Inhibitors of miR-21 are currently undergoing clinical trials. Our aim was to explore mechanisms of how miR-21 inhibition attenuates fibrosis using a proteomics approach. Methods and results Primary murine cardiac fibroblasts (CF) were transfected with miR-21 mimics and inhibitors. In line with previous reports, miR-21 overexpression increased CF proliferation (n=4 vs. 4; p Conclusions This study reports a previously unrecognised effect of miR-21 inhibition on platelet TGF-β1 release. This novel systemic effect of miR-21 on platelets may contribute to the anti-fibrotic effects of miR-21 inhibitors.

We would like to thank Dr Grosser et al1 for their continued interest in our work. While we may not agree with their arguments or their opinions of our study,2 we welcome debate in this extremely important area. We take this opportunity to address the points that they raise. First, although urine may well be a convenient compartment within which to measure markers of prostacyclin and thromboxane A2, numerous observations,3–10 of which ours is only the latest,2 indicate that they poorly reflect production within the circulation and the reactivities of endothelial cells and platelets. Second, the literature that Grosser et al1 cite is somewhat selective and in places inaccurate. For example, reference 3,11 which the authors cite to substantiate the statement “Most insights into the in vivo biology and pharmacology of the prostaglandin pathway have derived from the …

Platelets are important players in thrombosis and haemostasis with their function being modulated by mediators in the blood and the vascular wall. Among these, eicosanoids can both stimulate and inhibit platelet reactivity. Platelet Cyclooxygenase (COX)‐1‐generated Thromboxane (TX)A2 is the primary prostanoid that stimulates platelet aggregation; its action is counter‐balanced by prostacyclin, a product of vascular COX. Prostaglandin (PG)D2, PGE2 and 12‐hydroxyeicosatraenoic acid (HETE), or 15‐HETE, are other prostanoid modulators of platelet activity, but some also play a role in carcinogenesis. Aspirin permanently inhibits platelet COX‐1, underlying its anti‐thrombotic and anti‐cancer action. While the use of aspirin as an anti‐cancer drug is increasingly encouraged, its continued use in addition to P2Y12 receptor antagonists for the treatment of cardiovascular diseases is currently debated. Aspirin not only suppresses TXA2 but also prevents the synthesis of both known and unknown antiplatelet eicosanoid pathways, potentially lessening the efficacy of dual antiplatelet therapies. Linked Articles This article is part of a themed section on Eicosanoids 35 years from the 1982 Nobel: where are we now? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.8/issuetoc

The current guidelines following an acute coronary syndrome recommend dual-antiplatelet therapy (DAPT) (aspirin plus a P2YTo explore how exercise-induced improvements in vascular and platelet function affect the efficacy of DAPT, in a cross-sectional study of men with different physical activity levels (training status).A total of 42 healthy, normal-weight, middle-aged men were divided into 3 groups: untrained, moderately trained and well-trained. Their platelet reactivity (agonist-induced % aggregation) was investigated in platelet-rich plasma at rest and after inhibition with aspirin and ticagrelor and/or prostacyclin and nitric oxide added to the blood in vitro, and after physiological tests of vascular function; passive movement of the leg, flow-mediated dilation and one-leg knee-extensor exercise. Vascular function of the femoral artery (changes in arterial blood flow) was assessed by ultrasound Doppler.Platelets from the well-trained subjects had lower basal reactivity, a higher sensitivity to the anti-aggregatory effects of prostacyclin and were more potently inhibited by DAPT compared to the untrained subjects. The moderately trained and well-trained subjects had a superior vascular function compared to untrained subjects, and their platelets were more inhibited by the passive movement, flow-mediated dilation and one-leg knee-extensor exercise.A habitually active lifestyle leads to an increased platelet sensitivity to pharmacological and physiological platelet inhibitors. We suggest that physical activity habits (training status) should be considered when personalizing and optimizing antithrombotic treatment strategies.

Testing of platelet function is central to the cardiovascular phenotyping of genetically modified mice. Traditional platelet function tests have been developed primarily for testing human samples and the volumes required make them highly unsuitable for the testing of mouse platelets. This limits research in this area. To address this problem, we have developed a miniaturized whole blood aggregometry assay, based on a readily accessible 96-well plate format coupled with quantification of single platelet depletion by flow cytometric analysis. Using this approach, we observed a concentration-dependent loss of single platelets in blood exposed to arachidonic acid, collagen, U46619 or protease activated receptor 4 activating peptide. This loss was sensitive to well-established antiplatelet agents and genetic manipulation of platelet activation pathways. Observations were more deeply analyzed by flow cytometric imaging, confocal imaging, and measurement of platelet releasates. Phenotypic analysis of the reactivity of platelets taken from mice lacking intercellular adhesion molecule (ICAM)-1 identified a marked decrease in fibrinogen-dependent platelet-monocyte interactions, especially under inflammatory conditions. Such findings exemplify the value of screening platelet phenotypes of genetically modified mice and shed further light upon the roles and interactions of platelets in inflammation.

Hydrogen sulfide (H2S) is a gaseous meditator that has various physiological and pathophysiological roles in the body. It has been shown to be an important mediator of gastrointestinal (GI) mucosal defense and contributes significantly to repair of damage and resolution of inflammation. Synthesis of H2S increases markedly after mucosal injury, and inhibition of H2S in such circumstances leads to delayed healing and exacerbated inflammation. The beneficial effects of H2S may be attributable to its ability to elevate mucosal blood flow, prevent leukocyte-endothelial adhesion, reduce oxidative stress, and stimulate angiogenesis. The use of H2S-donating agents and inhibitors of the key enzymes contributing to H2S synthesis have provided strong evidence for the importance of H2S in enhancing mucosal resistance to damage, as well as modulating inflammation and repair. In recent years, significant evidence has been generated to support the notion that these positive aspects of H2S can be exploited in drug design, particularly for arthritis, inflammatory bowel disease, and colon cancer chemoprevention. Thus novel H2S-based therapies have been shown to be effective anti-inflammatories that can promote the resolution of inflammation and accelerate the healing of GI ulcers. Encouraging results have already been seen experimentally with a mesalamine derivative and with H2S-releasing derivatives of nonsteroidal anti-inflammatory drugs.

Inherited defects of platelet function disorders are rare and difficult to diagnose due to lack of standardized platelet tests. An aspirin-like platelet defect is characterised by reduced thromboxane A2 (TXA2) signalling due to a defect in the arachidonic acid (AA) pathway in platelets. Patients with aspirin-like defect present with mild to moderate bleeding symptoms and impaired platelet aggregation responses to AA and ADP. This is similar to the irreversible effect of aspirin on platelets, which is mediated through inhibition of prostaglandin H synthase-1 also known as cyclooxygenase-1 (PTGS1/COX1). We for the first time report platelet function disorders due to autosomal recessive inheritance of variants in PTGS1. In a total of 3563 cases with bleeding disorders, comprising 1169 whole genome sequenced probands of the BRIDGE-BPD study and 2394 panel sequenced index cases of the ThromboGenomics cohort, we identified 15 unrelated cases of each cohort with an aspirin-like platelet function defect. Two of these cases had rare a variants in PTGS1, the gene encoding COX-1, which catalyses the conversion of arachidonic acid to prostaglandin H2. The first case presented with epistaxis and peri-operative bleeding. She had reduced platelet aggregation responses to arachidonic acid, ADP, collagen and epinephrine. Incubation of control blood with collagen resulted in enhanced levels of thromboxane B2, PGD2, PGE2, 11-HETE and 15-HETE which was absent in the index case.We identified a homozygous missense variant in PTGS1, p.Trp322Ser with a Combined Annotation Dependant Depletion Score (CADD) of 31.0. This variant was absent from GnomAD. The variant co-segregated in an autosomal recessive inheritance mode, with aspirin-like defect phenotype in the seven family members who were investigated. PTGS1 was not expressed on the platelets by western blot. Immunophenotyping demonstrated absence on the platelet surface but presence on neutrophils. The second case of the presented with menorrhagia, nosebleeds, easy bleeding and bruising. She had reduced aggregation responses to arachidonic acid, ADP, collagen and epinephrine. We identified two variants in cis: a splice-donor variant (g. 125133553 T>A), CADD 24.3; and an upstream non-coding variant (g. 125132069 C>G), CADD 16.63. The frequency of these variants were respectively; 1.7 x 10-5 and 8 x 10-3 in GnomAD. Platelet RNA and protein expression studies in the propositus revealed alternative splicing with the generation of a smaller protein due the splice variant. In contrast, the non coding variant had no effect on promoter or enhancer activity and therefore, is likely benign. In this case, the mode of inheritance is autosomal dominant with a dominant negative effect, which has been reported previously. For the other 13 cases of the Bridge-BPD study we also interrogated the non-coding space and interactors in the arachidonic acid pathway, none of which had genetic variants explaining the phenotype. For the 15 ThromboGenomics cohort cases because they were sequenced on targeted platform similar investigations could not take place. These cases could have a non inherited cause for the platelet defect or it is also permissible that variation in a hitherto undefined pathways unique to individual cases might be causal. In conclusion, we for the first time report autosomal recessive inheritance of variants in PTGS1 as cause for a rare inherited bleeding disorder. The effect of the mutation are selective loss of expression of PTGS1 within platelets and decreased enzyme function. Two previous reports demonstrated autosomal dominant inheritance. The first demonstrated autosomal dominant inheritance of variants in PTGS1 as modifier in a well characterized family with haemophilia A and platelet function disorder (Nance et al JTH 2016). The second reported rare heterozygous variants in PTGS1 in two cases with a bleeding tendency which was not further specified in the report (Bastida et al Haematologica 2018). Disclosures Laffan: Pfizer: Honoraria; Roche: Consultancy, Speakers Bureau.

Nonsteroidal anti-inflammatory drugs have been shown to reduce the incidence of gastrointestinal cancers, but the propensity of these drugs to cause ulcers and bleeding limits their use. H2S has been shown to be a powerful cytoprotective and anti-inflammatory substance in the digestive system. This study explored the possibility that a H2S-releasing nonsteroidal anti-inflammatory drug (ATB-346) would be effective in a murine model of hereditary intestinal cancer (APCMin+ mouse) and investigated potential mechanisms of action via transcriptomics analysis. Daily treatment with ATB-346 was significantly more effective at preventing intestinal polyp formation than naproxen. Significant beneficial effects were seen with a treatment period of only 3–7 days, and reversal of existing polyps was observed in the colon. ATB-346, but not naproxen, significantly decreased expression of intestinal cancer-associated signaling molecules (cMyc, β-catenin). Transcriptomic analysis identified 20 genes that were up-regulated in APCMin+ mice, 18 of which were reduced to wild-type levels by one week of treatment with ATB-346. ATB-346 is a novel, gastrointestinal-sparing anti-inflammatory drug that potently and rapidly prevents and reverses the development of pre-cancerous lesions in a mouse model of hereditary intestinal tumorigenesis. These effects may be related to the combined effects of suppression of cyclooxygenase and release of H2S, and correction of most of the APCMin+-associated alterations in the transcriptome. ATB-346 may represent a promising agent for chemoprevention of tumorigenesis in the GI tract and elsewhere.

BACKGROUND AND PURPOSE Pre-menopausal females have a lower incidence of cardiovascular disease compared with age-matched males, implying differences in the mechanisms and pathways regulating vasoactivity. In small arteries, myogenic tone (constriction in response to raised intraluminal pressure) is a major determinant of vascular resistance. Endothelium-derived dilators, particularly NO, tonically moderate myogenic tone and, because the endothelium is an important target for female sex hormones, we investigated whether NO-mediated moderation of myogenic tone differed between the sexes. EXPERIMENTAL APPROACH Pressure–diameter or relaxation concentration–response curves to the NO donor spermine-NO or soluble guanylate cyclase (sGC) stimulation (BAY41-2272) were constructed before and following drug intervention in murine mesenteric resistance arteries. Hypotensive responses to activators of the NO-sGC pathway were determined. Quantitative PCR and Western blotting were used for expression analysis. KEY RESULTS NO synthase inhibition enhanced myogenic tone of arteries of both sexes while block of endothelium-derived hyperpolarizing factor (EDHF) enhanced responses in arteries of females only. Spermine-NO concentration-dependently relaxed mesenteric arteries isolated from either sex. However, while inhibition of sGC activity attenuated responses of arteries from male mice only, endothelial denudation attenuated responses of arteries from females only. BAY41-2272 and spermine-NO-induced vasodilatation and hypotension were greater in males than in females. CONCLUSIONS AND IMPLICATIONS NO moderated myogenic tone in arteries of male mice by a sGC-dependent pathway while EDHF was the predominant endothelial regulator in arteries of females. This is a potentially important sexual dimorphism in NO-mediated reactivity and further implicates EDHF as the predominant endothelial vasodilator in female resistance arteries.

Cyclooxygenase-2 (COX-2) is an inducible enzyme that drives inflammation and is the therapeutic target for widely used nonsteroidal antiinflammatory drugs (NSAIDs). However, COX-2 is also constitutively expressed, in the absence of overt inflammation, with a specific tissue distribution that includes the kidney, gastrointestinal tract, brain, and thymus. Constitutive COX-2 expression is therapeutically important because NSAIDs cause cardiovascular and renal side effects in otherwise healthy individuals. These side effects are now of major concern globally. However, the pathways driving constitutive COX-2 expression remain poorly understood. Here we show that in the kidney and other sites, constitutive COX-2 expression is a sterile response, independent of commensal microorganisms and not associated with activity of the inflammatory transcription factor NF-κB. Instead, COX-2 expression in the kidney but not other regions colocalized with nuclear factor of activated T cells (NFAT) transcription factor activity and was sensitive to inhibition of calcineurin-dependent NFAT activation. However, calcineurin/NFAT regulation did not contribute to constitutive expression elsewhere or to inflammatory COX-2 induction at any site. These data address the mechanisms driving constitutive COX-2 and suggest that by targeting transcription it may be possible to develop antiinflammatory therapies that spare the constitutive expression necessary for normal homeostatic functions, including those important to the cardiovascular-renal system.

In our recent report of the optical multichannel (optimul) method [1], we demonstrated a viable, standardised approach to platelet aggregometry. We proposed that this method of platelet reactivity ...

Summary. Background: Aspirin and antagonists of platelet ADP P2Y12 receptors are often coprescribed for protection against thrombotic events. However, blockade of platelet P2Y12 receptors can inhibit thromboxane A2 (TXA2)-dependent pathways of platelet activation independently of aspirin. Objectives: To assess in vitro whether aspirin adds additional antiaggregatory effects to strong P2Y12 receptor blockade. Methods: With the use of platelet-rich plasma from healthy volunteers, determinations were made in 96-well plates of platelet aggregation, TXA2 production and ADP/ATP release caused by ADP, arachidonic acid, collagen, epinephrine, TRAP-6 amide and U46619 (six concentrations of each) in the presence of prasugrel active metabolite (PAM; 0.1–10 μmol L−1), aspirin (30 μmol L−1), PAM + aspirin or vehicle. Results: PAM concentration-dependently inhibited aggregation; for example, aggregation in response to all concentrations of ADP and U46619 was inhibited by ≥ 95% by PAM at > 3 μmol L−1. In further tests of PAM (3 μmol L−1), aspirin (30 μmol L−1) and PAM + aspirin, aspirin generally failed to produce more inhibition than PAM or additional inhibition to that caused by PAM. The antiaggregatory effects of PAM were associated with reductions in the platelet release of both TXA2 and ATP + ADP. Similar effects were found when either citrate or lepirudin were used as anticoagulants, and when traditional light transmission aggregometry was conducted at low stirring speeds. Conclusions: P2Y12 receptors are critical to the generation of irreversible aggregation through the TXA2-dependent pathway. As a result, strong P2Y12 receptor blockade alone causes inhibition of platelet aggregation that is little enhanced by aspirin. The clinical relevance of these observations remains to be determined.

Juvenile idiopathic arthritis (JIA) has an incidence that ranges from 1 to 22 per 100,000 children worldwide, with involvement of the temporomandibular joint (TMJ) in 17–87% of patients. Intraarticular corticosteroid injections are beneficial in the local treatment of JIA and of other types of arthritis. To describe and assess the accuracy of an US-guided technique for visualization of needle placement within the TMJ in children. Between January 2000 and November 2007, 180 TMJ injections were performed during 116 encounters in 83 children with arthritis (71 girls, 12 boys; mean age 12.0 years). Access was obtained under sterile conditions using US guidance (linear 15-MHz or curvilinear 8-MHz transducers) in a coronal plane, and confirmed with CT. To minimize radiation, a limited focused CT protocol was developed. A bilateral injection was performed in 65 encounters (57%). Twenty-three children had repeat TMJ injections. All injections were performed using US guidance. CT confirmation was used in 127/180 TMJs (70%). In those confirmed with CT, the needle tip was intra-articular in 91% of cases. Triamcinolone hexacetonide was used in 92% of injections and triamcinolone acetonide in 8%. One major complication was encountered (skin atrophy at the injection site). In our experience, TMJ injections using sonographic guidance is a safe, effective and accurate procedure.

Objective— Reduced antiplatelet drug efficacy occurs in conditions of increased platelet turnover, associated with increased proportions of drug-free, that is, uninhibited, platelets. Here, we detail mechanisms by which drug-free platelets promote platelet aggregation in the face of standard antiplatelet therapy. Approach and Results— To model standard antiplatelet therapy, platelets were treated in vitro with aspirin, the P2Y 12 receptor blocker prasugrel active metabolite, or aspirin plus prasugrel active metabolite. Different proportions of uninhibited platelets were then introduced. Light transmission aggregometry analysis demonstrated clear positive associations between proportions of drug-free platelets and percentage platelet aggregation in response to a range of platelet agonists. Using differential platelet labeling coupled with advanced flow cytometry and confocal imaging we found aggregates formed in mixtures of aspirin-inhibited platelets together with drug-free platelets were characterized by intermingled platelet populations. This distribution is in accordance with the ability of drug-free platelets to generate thromboxane A 2 and so drive secondary platelet activation. Conversely, aggregates formed in mixtures of prasugrel active metabolite–inhibited or aspirin plus prasugrel active metabolite–inhibited platelets together with drug-free platelets were characterized by distinct cores of drug-free platelets. This distribution is consistent with the ability of drug-free platelets to respond to the secondary activator ADP. Conclusions— These experiments are the first to image the interactions of inhibited and uninhibited platelets in the formation of platelet aggregates. They demonstrate that a general population of platelets can contain subpopulations that respond strikingly differently to overall stimulation of the population and so act as the seed for platelet aggregation.

Significance: There is a rapidly expanding body of evidence for important roles of hydrogen sulfide in protecting against tissue injury, reducing inflammation, and promoting repair. There is also growing evidence that H2S can be successfully exploited in drug development. Recent Advances: H2S synthesis and degradation are regulated in circumstances of inflammation and injury so as to promote repair and re-establish homeostasis. Novel H2S-releasing drugs exhibit enhanced anti-inflammatory and pro-restorative effects, while having reduced adverse effects in many tissues. Critical Issues: H2S is a pleiotropic mediator, having effects on many elements in the inflammatory cascade and promoting the resolution of inflammation and injury. It also contributes significantly to mucosal defence in the gastrointestinal tract, and in host defence against infection. There is strong evidence that novel, H2S-based therapeutics are safe and effective in animal models, and several are progressing through human trials. Future Directions: A better understanding of the physiological and pathophysiological roles of H2S continues to be restrained by the lack of simple, reliable methods for measurement of H2S synthesis, and the paucity of highly selective inhibitors of enzymes that participate in endogenous H2S synthesis. On the other hand, H2S donors show promise as therapeutics for several important indications. Antioxid. Redox Signal. 22, 398–410.

Rationale: Platelets shed microRNAs (miRNAs). Plasma miRNAs change on platelet inhibition. It is unclear whether plasma miRNA levels correlate with platelet function. Objective: To link small RNAs to platelet reactivity. Methods and Results: Next-generation sequencing of small RNAs in plasma revealed 2 peaks at 22 to 23 and 32 to 33 nucleotides corresponding to miRNAs and YRNAs, respectively. Among YRNAs, predominantly, fragments of RNY4 and RNY5 were detected. Plasma miRNAs and YRNAs were measured in 125 patients with a history of acute coronary syndrome who had undergone detailed assessment of platelet function 30 days after the acute event. Using quantitative real-time polymerase chain reactions, 92 miRNAs were assessed in patients with acute coronary syndrome on different antiplatelet therapies. Key platelet-related miRNAs and YRNAs were correlated with platelet function tests. MiR-223 ( r p =0.28; n=121; P =0.002), miR-126 ( r p =0.22; n=121; P =0.016), and other abundant platelet miRNAs and YRNAs showed significant positive correlations with the vasodilator-stimulated phosphoprotein phosphorylation assay. YRNAs, miR-126, and miR-223 were also among the small RNAs showing the greatest dependency on platelets and strongly correlated with plasma levels of P-selectin, platelet factor 4, and platelet basic protein in the population-based Bruneck study (n=669). A single-nucleotide polymorphism that facilitates processing of pri-miR-126 to mature miR-126 accounted for a rise in circulating platelet activation markers. Inhibition of miR-126 in mice reduced platelet aggregation. MiR-126 directly and indirectly affects ADAM9 and P2Y 12 receptor expression. Conclusions: Levels of platelet-related plasma miRNAs and YRNAs correlate with platelet function tests in patients with acute coronary syndrome and platelet activation markers in the general population. Alterations in miR-126 affect platelet reactivity.

Introduction Aspirin acts as an anti-thrombotic drug by inhibiting platelet cyclooxygenase-1 (COX-1) and blocking the production of thromboxane A2, but it also has off-platelet actions that may limit its effectiveness. Since mice have very marked differences in aspirin pharmacokinetics they cannot be used to explore the effects of low dose aspirin in humans. To address these questions, we developed platelet COX-1 knockout mice (platelet-COX-1-/-) to recapitulate the effect of low-dose aspirin in humans. Methods and Results Cox-1fl/fl mice were paired with Pf4-Cre mice to delete COX-1 in the MK lineage. The absence of COX-1 in platelets was confirmed by Western blot analysis, confocal immunofluorescence microscopy and by analysing the distribution of the COX-1 signal and mass with an high-throughput confocal imaging system. The production of thromboxane B2 (TxB2) induced by collagen, A23187 and the PAR-4 activating peptide in platelet rich plasma and whole blood was significantly decreased in platelet-COX-1-/- mice. This effect was not further reduced by the addition of aspirin in vitro. Further eicosanomic analysis was performed using LC-MS/MS of samples from untreated and A23187-stimulated whole blood. 11- and 15-hydroxyeicosatetraenoic acid (HETE), alongside TxB2 formation were inhibited after platelet COX-1 deletion. Production of 12-HETE, coming from 12-lipoxygenase (LOX), and 5-HETE coming from leukocyte 5-LOX, was unchanged in platelet-COX-1-/- mice. To better dissect the effects of aspirin in vivo, we used a thrombosis model caused by ferric chloride injury in the mouse carotid artery. Platelet COX-1-/- mice, that recapitulate the effects of low-dose aspirin, formed unstable thrombi that were embolizing before producing a stable vessel occlusion. Interestingly, when a high dose of aspirin (10 mg/kg) was injected into the mice, the thrombus was stable and formed earlier than in the vehicle-treated mice. Conclusions We have produced the first platelet-COX-1-/- mouse and characterised its phenotype and eicosanomic profile. This model is allowing us to mechanistically dissect the platelet and non-platelet effects of aspirin and so better model the effects of clinically important anti-thrombotic therapy.

‘Dual antiplatelet therapy’, comprising aspirin and a P2Y12 receptor inhibitor, is firmly established for the secondary prevention of thrombotic events with the rationale that they inhibit thromboxane A2- (TxA2) and ADP-P2Y12-dependent pathways of platelet activation, respectively. We have recently reported that strong P2Y12 receptor blockade alone, however, can provide inhibition of platelet aggregation to a broad range of agonists that is not further enhanced by aspirin [1]. While the clinical relevance of these observations is unclear, we have speculated that administration of aspirin to individuals achieving sufficiently strong P2Y12 receptor blockade, has the potential to produce effects secondary to inhibition of cyclo-oxygenase at non-platelet sites, without providing additional antithrombotic activity [2]. The degree of P2Y12 pathway blockade that is achieved in clinical practice, however, is quite variable [3], reflecting both the choice of drug and large inter-individual differences in drug metabolism [4]. Here, we have extended our previous observations of the interactions between aspirin and strong P2Y12 blockade [1] by considering what additional anti-aggregatory effects aspirin provides when only partial P2Y12 blockade is achieved, which may better reflect the clinical reality of these drugs. We measured aggregation responses of platelet-rich plasma (PRP), using 96-well plate light transmission aggregometry, as previously described [1]. Blood was collected by venepuncture into tri-sodium citrate (0.32% final) from healthy volunteers who had abstained from non-steroid anti-inflammatory drug consumption for 14 days. To model the effects of P2Y12 blockade and cyclo-oxygenase inhibition in vitro, PRP was incubated with the irreversible thienopyridine P2Y12 inhibitor, prasgurel-active metabolite (PAM; 0.1–10 μmol L−1), the reversible, cyclo-pentyl-triazolo-pyrimidine P2Y12 antagonist, ticagrelor (0.1–10 μmol L−1) and/or aspirin (1–100 μmol L−1) for 30 min at 37 °C before addition of the agonist. Additional methodological details are provided as online supplementary information. Using this approach we determined the inhibitory potencies of ticagrelor, PAM and aspirin against aggregations induced by ADP (0.625–20 μmol L−1), the thromboxane-mimetic U46619 (0.1–30 μmol L−1) and arachidonic acid (0.1–1 mmol L−1). Both ticagrelor and PAM caused concentration-dependent inhibition of aggregations induced by ADP (Fig. S1), with ticagrelor displaying greater potency than PAM (log IC50 values for inhibition of aggregation to 20 μmol L−1 ADP: ticagrelor, −6.46; PAM, −5.64). Notably, the potency of ticagrelor, but not PAM, varied with the concentration of ADP (e.g. log IC50 values for inhibition of aggregation to 2.5 μmol L−1 ADP: ticagrelor, −7.05; PAM, −5.63). Aspirin, at concentrations up to 100 μmol L−1, was without significant effect upon ADP-induced aggregations. Ticagrelor and PAM, but not aspirin, produced complete, concentration-dependent inhibition of platelet aggregations induced by U46619 (Fig. S2) with similar potency as for inhibition of ADP-induced aggregations (log IC50 values for inhibition of aggregation to 30 μmol L−1 U46619; ticagrelor, −6.24; PAM, −5.25). This is consistent with earlier reports that the second, irreversible wave of platelet aggregation that follows TP receptor activation is dependent upon platelet-derived ADP acting upon platelet P2Y12 receptors [5]. Ticagrelor and PAM, as well as aspirin, also produced complete, concentration-dependent inhibitions of platelet aggregations induced by AA (Fig. S3; log IC50 values for inhibition of aggregation to 1 mmol L−1 AA: ticagrelor, −6.88; PAM, −6.00; aspirin, −5.20). When the production of TxA2 accompanying platelet aggregation induced by AA was measured (by immunoassay for the levels of TxB2), ticagrelor, PAM and aspirin were all found to cause concentration-dependent reductions in TxA2 formation (Fig. S3; log IC50 values for inhibition of aggregation to 1 mmol L−1 AA: ticagrelor, −6.88; PAM, −5.985; aspirin, −5.51). This is in agreement with our early findings [1, 6], and indicates that P2Y12 receptors are important in supporting both the activation mechanisms of platelets that drive TxA2 formation and pathways downstream of the TP receptor. To explore further the interactions between P2Y12 receptors and the TxA2 system in platelets, we examined the effect of aspirin on aggregation induced by a range of agonists in the presence of concentrations of ticagrelor or PAM producing different degrees of partial P2Y12 blockade. From the inhibitor curves to ADP described above, concentrations of ticagrelor showing approximate IC5 (0.03 μmol L−1), IC10 (0.1 μmol L−1), IC50 (0.3 μmol L−1) and IC90 effects (3 μmol L−1) were combined with 30 μmol L−1 aspirin, a concentration approximately equivalent to the peak plasma levels following ingestion of a 75–100 mg dose of aspirin. In these experiments, responses to AA (Fig. 1A) were found to be completely inhibited by ticagrelor at the higher two concentrations (representing ∼IC50 and IC90 for ADP-induced aggregation) without the need for aspirin. The lower two concentrations of ticagrelor (representing ∼IC5 and IC10 for ADP-induced aggregation) also produced substantial, but incomplete, inhibitions (Table S1). Ticagrelor also inhibited aggregations induced by ADP, collagen, epinephrine, the PAR-1 activating peptide, TRAP-6 (SFLLRN-amide) and U46619, in a concentration-dependent manner (Fig. 1). Figure 1 Concentration-response curves for the inhibition by combinations of ticagrelor (0.3 or 3 μmol L−1) and aspirin (30 μmol L−1) of platelet aggregations induced by (A) arachidonic acid (AA), (B) ADP, (C) collagen, (D) epinephrine, ... When applied alone, aspirin inhibited aggregations induced by AA (Fig. 1A), collagen (Fig. 1C) and epinephrine (Fig. 1D), and showed a weak effect against ADP (Fig. 1B) but did not alter aggregations induced by TRAP-6 (Fig. 1E) or U46619 (Fig. 1F). In contrast, aspirin did augment the anti-aggregatory effects of the lower three concentrations of ticagrelor (achieving incomplete P2Y12 inhibition) against both collagen (Fig. 1C) and epinephrine (Fig. 1D). In the presence of the highest tested concentration of ticagrelor (3 μmol L−1; ∼IC90 for ADP-induced aggregation), aspirin provided no additional anti-aggregatory effects to those of ticagrelor against aggregations to any agonist (Fig. 1). In agreement with earlier experiments, production of TxA2 induced by either AA (Fig. 1G) or collagen (Fig. 1H) was partially inhibited by 0.3 μmol L−1 ticagrelor (∼IC50 for ADP-induced aggregation) and abolished by 3 μmol L−1 ticagrelor (∼IC90 for ADP-induced aggregation). We have previously reported that TxA2 production in response to epinephrine is inhibited by P2Y12 blockade in the same manner for AA and collagen [1]. Aspirin (30 μmol L−1) either alone or in combination with ticagrelor also completely inhibited TxA2 production to either agonist (Fig. 1G,H). A similar pattern of results was obtained using equivalent inhibitory concentrations of PAM in place of ticagrelor (Table S2), and when the concentration of aspirin was increased to 120 μmol L−1 (Tables S1 and S2). These studies show that ticagrelor and PAM inhibit platelet aggregation induced by a range of platelet agonists through a mechanism consistent with blockade of platelet P2Y12 receptors and that ticagrelor is more potent than PAM in this regard. As well as inhibiting aggregation following from direct activation of P2Y12 receptors by the addition of exogenous ADP, ticagrelor and PAM inhibited aggregations resulting from stimulation of platelets with AA, a response which is well characterized as being TxA2 dependent. In addition to inhibiting platelet responses to endogenously produced TxA2, ticagrelor and PAM also inhibited the production of TxA2 by platelets [6]. Interestingly, ADP itself is a poor stimulus for TxA2 production [1], suggesting that released ADP, acting on the P2Y12 receptor, acts to potentiate the stimulation of TxA2 synthesis by other signaling pathways activated in parallel. These results are consistent with the idea that whereas aspirin may inhibit just the TxA2-dependent pathway of platelet activation, ticagrelor and PAM can inhibit both the ADP-P2Y12-dependent and the TxA2-dependent pathways of platelet aggregation. The observation that aspirin adds anti-aggregatory effects to partial, but not complete, P2Y12 receptor blockade, further supports this idea. Taken together these results demonstrate that rather than ADP-P2Y12 and TxA2 pathways acting independently, the TxA2-dependent pathway is dependent upon the ADP-P2Y12 pathway both for the production of TxA2 and fundamentally for the irreversible aggregation that follows activation of TP receptors. If these data accurately model the situation in vivo, this may have important implications for the use of dual antiplatelet therapy using potent P2Y12 antagonists in clinical practice [2]. For example, one could postulate that addition of aspirin could produce side-effects secondary to inhibition of cyclo-oxygenase at non-platelet sites, as has recently become apparent for non-steroid anti-inflammatory drugs, while providing little additional anti-aggregatory effect [7, 8]. Clearly, the validity of this hypothesis remains to be determined by clinical studies.