Nicholas S Kirkby

Imperial College London, Londinium, England, United Kingdom

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Publications (28)175.98 Total impact

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    ABSTRACT: -Cardiovascular side effects associated with cyclo-oxygenase-2 inhibitor drugs dominate clinical concern. Cyclo-oxygeanse-2 is expressed in the renal medulla where inhibition causes fluid retention and increased blood pressure. However, the mechanisms linking cyclo-oxygeanse-2 inhibition and cardiovascular events are unknown and no biomarkers have been identified. -Transcriptome analysis of wild-type and cyclo-oxygenase-2(-/-) mouse tissues revealed 1 gene altered in heart and aorta but >1000 genes in the renal medulla including those regulating the endogenous NO synthase inhibitors ADMA and L-NMMA; Cyclo-oxygeanse-2(-/-) mice had increased plasma levels of ADMA and L-NMMA and reduced endothelial NO responses. These genes and methylarginines were not similarly altered in mice lacking prostacyclin receptors (IP(-/-)). Wild-type mice or human volunteers taking cyclo-oxygeanse-2 inhibitors also showed increased plasma ADMA. Endothelial NO is cardio protective, reducing thrombosis and atherosclerosis. Consequently, increased ADMA is associated with cardiovascular disease. Thus, our study identifies ADMA as a biomarker and mechanistic bridge between renal cyclo-oxygenase-2 inhibition and systemic vascular dysfunction with non-steroidal anti-inflammatory drug usage. -We identify the endogenous eNOS inhibitor ADMA as a biomarker and mechanistic bridge between renal COX-2 inhibition and systemic vascular dysfunction.
    Circulation 12/2014; · 15.20 Impact Factor
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    ABSTRACT: Endothelial cells form a highly specialised lining of all blood vessels where they provide an anti-thrombotic surface on the luminal side and protect the underlying vascular smooth muscle on the abluminal side. Specialised functions of endothelial cells include their unique ability to release vasoactive hormones and to morphologically adapt to complex shear stress. Stem cell derived-endothelial cells have a growing number of applications and will be critical in any organ regeneration programme. Generally endothelial cells are identified in stem cell studies by well-recognised markers such as CD31. However, the ability of stem cell-derived endothelial cells to release vasoactive hormones and align with shear stress has not been studied extensively. With this in mind, we have compared directly the ability of endothelial cells derived from a range of stem cell sources, including embryonic stem cells (hESC-EC) and adult progenitors in blood (blood out growth endothelial cells, BOEC) with those cultured from mature vessels, to release the vasoconstrictor peptide endothelin (ET)-1, the cardioprotective hormone prostacyclin, and to respond morphologically to conditions of complex shear stress. All endothelial cell types, except hESC-EC, released high and comparable levels of ET-1 and prostacyclin. Under static culture conditions all endothelial cell types, except for hESC-EC, had the typical cobblestone morphology whilst hESC-EC had an elongated phenotype. When cells were grown under shear stress endothelial cells from vessels (human aorta) or BOEC elongated and aligned in the direction of shear. By contrast hESC-EC did not align in the direction of shear stress. These observations show key differences in endothelial cells derived from embryonic stem cells versus those from blood progenitor cells, and that BOEC are more similar than hESC-EC to endothelial cells from vessels. This may be advantageous in some settings particularly where an in vitro test bed is required. However, for other applications, because of low ET-1 release hESC-EC may prove to be protected from vascular inflammation.
    Biochemical and Biophysical Research Communications 11/2014; · 2.28 Impact Factor
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    ABSTRACT: Up to 1% of the general population have mild bleeding disorders, but these are often poorly characterized, particularly with regard to the roles of platelets. Here we have compared the usefulness of Optimul, a 96-well-plate-based assay of seven distinct pathways of platelet activation, to characterize inherited platelet defects in comparison to light transmission aggregometry (LTA). Using Optimul and LTA, concentration-response curves were generated for arachidonic acid, ADP, collagen, epinephrine, TRAP-6, U46619, and ristocetin in samples from (i) healthy volunteers (n=50), (ii) healthy volunteers treated with antiplatelet agents in vitro (n=10), and (iii) patients with bleeding of unknown origin (n=65). The assays gave concordant results in 82% of cases (κ=0.62, p<0.0001). Normal platelet function results were particularly predictive (sensitivity 94%, negative predictive value 91%) whereas a positive result was not always substantiated by LTA (specificity 67%, positive predictive value 77%). The Optimul assay was significantly more sensitive at characterizing defects in the thromboxane pathway, which nonetheless presented with normal responses with LTA. The Optimul assay is sensitive to mild platelet defects, could be used as a rapid screening assay in patients presenting with bleeding symptoms, and detects changes in platelet function more readily than LTA. This study is registered at www.isrctn.org, identifier: ISRCTN 77951167.
    Blood 01/2014; · 9.78 Impact Factor
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    ABSTRACT: Cyxlo-oxygenase (COX)-2 inhibitors, including traditional nonsteroidal anti-inflammatory drugs (NSAIDs) are associated with increased cardiovascular side effects, including myocardial infarction. We and others have shown that COX-1 and not COX-2 drives vascular prostacyclin in the healthy cardiovascular system, re-opening the question of how COX-2 might regulate cardiovascular health. In diseased, atherosclerotic vessels, the relative contribution of COX-2 to prostacyclin formation is not clear. Here we have used apoE-/-/COX-2-/- mice to show that, whilst COX-2 profoundly limits atherosclerosis, this protection is independent of local prostacyclin release. These data further illustrate the need to look for new explanations, targets and pathways to define the COX/NSAID/cardiovascular risk axis. Gene expression profiles in tissues from apoE-/-/COX-2-/- mice showed increased lymphocyte pathways that were validated by showing increased T-lymphocytes in plaques and elevated plasma Th1-type cytokines. In addition, we identified a novel target gene, rgl1, whose expression was strongly reduced by COX-2 deletion across all examined tissues. This study is the first to demonstrate that COX-2 protects vessels against atherosclerotic lesions independently of local vascular prostacyclin and uses systems biology approaches to identify new mechanisms relevant to development of next generation NSAIDs.
    PLoS ONE 01/2014; 9(6):e98165. · 3.53 Impact Factor
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    ABSTRACT: Evidence is increasing of a link between interferon (IFN) and pulmonary arterial hypertension (PAH). Conditions with chronically elevated endogenous IFNs such as systemic sclerosis (SSc) are strongly associated with PAH. Furthermore, therapeutic use of type I IFN is associated with PAH. This was recognised at the 2013 World Symposium on Pulmonary Hypertension where the urgent need for research into this was highlighted. To explore the role of type I IFN in PAH. Cells were cultured using standard approaches. Cytokines were measured by ELISA. Gene and protein expression were measured using RT-PCR, Western blotting and immunohistochemistry. The role of type I IFN in PAH in vivo was determined using type I IFN receptor knock out (IFNAR1(-/-)) mice. Human lung cells responded to types I and II but not III IFN correlating with relevant receptor expression. Type I, II and III IFN levels were elevated in serum of SSc-PAH patients. Serum IP10 and ET-1 were raised and strongly correlated together. IP10 correlated positively with pulmonary haemodynamics and serum brain natriuretic peptide and negatively with 6-minute walk test and cardiac index. Endothelial cells grown out of the blood of PAH patients were more sensitive to the effects of type I IFN than cells from healthy donors. PAH lung demonstrated increased IFNAR1 protein levels. IFNAR1(-/-) mice were protected from the effects of hypoxia on the right heart, vascular remodelling and raised serum ET-1 levels. These data indicate that type I IFN, via an action of IFNAR1, mediates PAH.
    Circulation Research 12/2013; · 11.86 Impact Factor
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    ABSTRACT: Homocysteine is metabolized to methionine by the action of 5,10 methylenetetrahydrofolate reductase (MTHFR). Alternatively, by the transulfuration pathway, homocysteine is transformed to hydrogen sulphide (H2S), through multiple steps involving cystathionine β-synthase and cystathionine γ-lyase. Here we have evaluated the involvement of H2S in the thrombotic events associated with hyperhomocysteinemia. To this purpose we have used platelets harvested from healthy volunteers or patients newly diagnosed with hyperhomocysteinemia with a C677T polymorphism of the MTHFR gene (MTHFR++). NaHS (0.1-100 µM) or l-cysteine (0.1-100 µM) significantly increased platelet aggregation harvested from healthy volunteers induced by thrombin receptor activator peptide-6 amide (2 µM) in a concentration-dependent manner. This increase was significantly potentiated in platelets harvested from MTHFR++ carriers, and it was reversed by the inhibition of either cystathionine β-synthase or cystathionine γ-lyase. Similarly, in MTHFR++ carriers, the content of H2S was significantly higher in either platelets or plasma compared with healthy volunteers. Interestingly, thromboxane A2 production was markedly increased in response to both NaHS or l-cysteine in platelets of healthy volunteers. The inhibition of phospholipase A2, cyclooxygenase, or blockade of the thromboxane receptor markedly reduced the effects of H2S. Finally, phosphorylated-phospholipase A2 expression was significantly higher in MTHFR++ carriers compared with healthy volunteers. In conclusion, the H2S pathway is involved in the prothrombotic events occurring in hyperhomocysteinemic patients.
    Proceedings of the National Academy of Sciences 09/2013; · 9.81 Impact Factor
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    ABSTRACT: Circulating platelets are constantly exposed to nitric oxide (NO) released from the vascular endothelium. This NO acts to reduce platelet reactivity, and in so doing blunts platelet aggregation and thrombus formation. For successful hemostasis, platelet activation and aggregation must occur at sites of vascular injury despite the constant presence of NO. As platelets aggregate, they release secondary mediators that drive further aggregation. Particularly significant among these secondary mediators is ADP, which, acting through platelet P2Y12 receptors, strongly amplifies aggregation. Platelet P2Y12 receptors are the targets of very widely used antithrombotic drugs such as clopidogrel, prasugrel, and ticagrelor. Here we show that blockade of platelet P2Y12 receptors dramatically enhances the antiplatelet potency of NO, causing a 1,000- to 100,000-fold increase in inhibitory activity against platelet aggregation and release reactions in response to activation of receptors for either thrombin or collagen. This powerful synergism is explained by blockade of a P2Y12 receptor-dependent, NO/cGMP-insensitive phosphatidylinositol 3-kinase pathway of platelet activation. These studies demonstrate that activation of the platelet ADP receptor, P2Y12, severely blunts the inhibitory effects of NO. The powerful antithrombotic effects of P2Y12 receptor blockers may, in part, be mediated by profound potentiation of the effects of endogenous NO.
    Proceedings of the National Academy of Sciences 09/2013; · 9.81 Impact Factor
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    ABSTRACT: Cyclooxygenase 2 (COX)-2 is induced by bacterial and viral infections and has complex, poorly understood roles in anti-pathogen immunity. Here, we use a knock-in luciferase reporter model to image Cox2 expression across a range of tissues in mice following treatment with the either the prototypical bacterial pathogen-associated molecular pattern (PAMP), LPS, which activates Toll-like receptor (TLR)4, or with poly(I:C), a viral PAMP, which activates TLR3. LPS induced Cox2 expression in all tissues examined. In contrast, poly(I:C) elicited a milder response, limited to a subset of tissues. A panel of cytokines and interferons was measured in plasma of wild-type, Cox1(-/-) and Cox2(-/-) mice treated with LPS, poly(I:C), MALP2 (TLR2/6), Pam3CSK4 (TLR2/1), R-848 (TLR7/8) or CpG ODN (TLR9), to establish whether/how each COX isoform modulates specific PAMP/TLR responses. Only LPS induced notable loss of condition in mice (inactivity, hunching, piloerection). However, all TLR agonists produced cytokine responses, many of which were modulated in specific fashions by Cox1 or Cox2 gene deletion. Notably we observed opposing effects of Cox2 gene deletion on the responses to the bacterial PAMP, LPS, and the viral PAMP, poly(I:C), consistent with the differing abilities of the PAMPs to induce Cox2 expression. Cox2 gene deletion limited the plasma IL-1β and interferon-γ responses and hypothermia produced by LPS. In contrast, in response to poly(I:C), Cox2(-/-) mice exhibited enhanced plasma interferon (IFNα,β,γ,λ) and related cytokine responses (IP-10,IL-12). These observations suggest that a COX-2 selective inhibitor, given early in infection, may enhance and/or prolong endogenous interferon responses, and thereby increase anti-viral immunity.
    Biochemical and Biophysical Research Communications 07/2013; · 2.28 Impact Factor
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    ABSTRACT: Inhibition of cyclooxygenase (COX)-2 increases cardiovascular deaths. Identifying a biomarker of COX-2 is desirable but difficult, since COX-1 and COX-2 ordinarily catalyze formation of an identical product, prostaglandin H2. When acetylated by aspirin, however, COX-2 (but not COX-1) can form 15(R)-HETE, which is metabolized to aspirin-triggered lipoxin (ATL), 15-epi-lipoxin A4. Here we have used COX-1- and COX-2-knockout mice to establish whether plasma ATL could be used as a biomarker of vascular COX-2 in vivo. Vascular COX-2 was low but increased by LPS (10 mg/kg; i.p). Aspirin (10 mg/kg; i.v.) inhibited COX-1, measured as blood thromboxane and COX-2, measured as lung PGE2. Aspirin also increased the levels of ATL in the lungs of LPS-treated wild-type C57Bl6 mice (vehicle: 25.5±9.3 ng/ml; 100 mg/kg: 112.0±7.4 ng/ml; P<0.05). Despite this, ATL was unchanged in plasma after LPS and aspirin. This was true in wild-type as well as COX-1(-/-) and COX-2(-/-) mice. Thus, in mice in which COX-2 has been induced by LPS treatment, aspirin triggers detectable 15-epi-lipoxin A4 in lung tissue, but not in plasma. This important study is the first to demonstrate that while ATL can be measured in tissue, plasma ATL is not a biomarker of vascular COX-2 expression.-Kirkby, N. S., Chan, M. V., Lundberg, M. H., Massey, K. A., Edmands, W. M. B., MacKenzie, L. S., Holmes, E., Nicolaou, A., Warner, T. D., Mitchell, J. A. Aspirin-triggered 15-epi-lipoxin A4 predicts cyclooxygenase-2 in the lungs of LPS-treated mice but not in the circulation: implications for a clinical test.
    The FASEB Journal 06/2013; · 5.70 Impact Factor
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    ABSTRACT: Acute renal failure (ARF) has high mortality and no effective treatment. Nitric oxide (NO) delivery represents a credible means of preventing the damaging effects of vasoconstriction, central to ARF, but design of drugs with the necessary renoselectivity is challenging. Here, we developed N-hydroxyguanidine NO donor drugs that were protected against spontaneous NO release by linkage to glutamyl adducts that could be cleaved by γ-glutamyl transpeptidase (γ-GT), found predominantly in renal tissue. Parent NO donor drug activity was optimized in advance of glutamyl adduct prodrug design. A lead compound that was a suitable substrate for γ-GT-mediated deprotection was identified. Metabolism of this prodrug to the active parent compound was confirmed in rat kidney homogenates, and the prodrug was shown to be an active vasodilator in rat isolated perfused kidneys (EC50 ∼50 μM). The data confirm that glutamate protection of N-hydroxyguanidines is an approach that might hold promise in ARF.
    Journal of Medicinal Chemistry 06/2013; · 5.61 Impact Factor
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    ABSTRACT: Cyclooxygenase (COX) is required for prostanoid (eg prostaglandin (PG)E2) production. Constitutive COX-1 and inducible COX-2 are implicated in lung diseases, such as idiopathic pulmonary fibrosis (IPF). Using lung fibroblasts from humans and wild type, COX-1-/- and COX-2 /- mice, we investigated how COX activity modulates cell growth and inflammatory responses induced by activators of Toll-like receptors (TLRs) 1-8. In mouse tissue, PGE2 release from fresh lung was COX-1 driven, in lung in culture (24hours) COX-1 and COX-2 driven, and from proliferating lung fibroblasts exclusively COX-2 driven. COX-2 limited proliferation in lung fibroblasts and both isoforms limited KC release induced by a range of TLR agonists. Less effect of COX was seen on TLR-induced IP-10 release. In human lung fibroblasts inhibition of COX with diclofenac was associated with increased release of IL-8 and IP-10. Our results may have implications for the treatment of IPF.
    Prostaglandins & other lipid mediators 06/2013; · 2.42 Impact Factor
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    ABSTRACT: Rationale: MicroRNA biomarkers are attracting considerable interest. Effects of medication, however, have not been investigated thus far. Objective: To analyse changes in plasma microRNAs in response to anti-platelet therapy. Methods and Results: Profiling for 377 microRNAs was performed in platelets, platelet microparticles, platelet-rich plasma, platelet-poor plasma and serum. Platelet-rich plasma showed markedly higher levels of microRNAs than serum and platelet-poor plasma. Few abundant platelet microRNAs, such as miR-24, miR-197, miR-191, and miR-223, were also increased in serum compared to platelet-poor plasma. In contrast, anti-platelet therapy significantly reduced microRNA levels. Using custom-made qPCR plates, 92 microRNAs were assessed in a dose-escalation study in healthy volunteers at four different time points: at baseline without therapy, at 1 week with 10mg prasugrel, at 2 weeks with 10mg prasugrel+75mg aspirin and at 3 weeks with 10mg prasugrel+300mg aspirin. Findings in healthy volunteers were confirmed by individual TaqMan qPCR assays (n=9). Validation was performed in an independent cohort of patients with symptomatic atherosclerosis (n=33) who received low dose aspirin at baseline. Plasma levels of platelet microRNAs, such as miR-223, miR-191 and others, i.e. miR-126 and miR-150, decreased upon further platelet inhibition. Conclusions: Our study demonstrated a substantial platelet contribution to the circulating microRNA pool and identified microRNAs responsive to anti-platelet therapy. It also highlights that anti-platelet therapy and preparation of blood samples could be confounding factors in case-control studies relating plasma microRNAs to cardiovascular disease.
    Circulation Research 01/2013; · 11.86 Impact Factor
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    ABSTRACT: There are two schools of thought regarding the cyclooxygenase (COX) isoform active in the vasculature. Using urinary prostacyclin markers some groups have proposed that vascular COX-2 drives prostacyclin release. In contrast, we and others have found that COX-1, not COX-2, is responsible for vascular prostacyclin production. Our experiments have relied on immunoassays to detect the prostacyclin breakdown product, 6-keto-PGF1α and antibodies to detect COX-2 protein. Whilst these are standard approaches, used by many laboratories, antibody-based techniques are inherently indirect and have been criticized as limiting the conclusions that can be drawn. To address this question, we measured production of prostanoids, including 6-keto-PGF1α, by isolated vessels and in the circulation in vivo using liquid chromatography tandem mass spectrometry and found values essentially identical to those obtained by immunoassay. In addition, we determined expression from the Cox2 gene using a knockin reporter mouse in which luciferase activity reflects Cox2 gene expression. Using this we confirm the aorta to be essentially devoid of Cox2 driven expression. In contrast, thymus, renal medulla, and regions of the brain and gut expressed substantial levels of luciferase activity, which correlated well with COX-2-dependent prostanoid production. These data are consistent with the conclusion that COX-1 drives vascular prostacyclin release and puts the sparse expression of Cox2 in the vasculature in the context of the rest of the body. In doing so, we have identified the thymus, gut, brain and other tissues as target organs for consideration in developing a new understanding of how COX-2 protects the cardiovascular system.
    PLoS ONE 01/2013; 8(7):e69524. · 3.53 Impact Factor
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    ABSTRACT: OBJECTIVE: Cryptogenic multifocal ulcerating stenosing enteritis (CMUSE) is an extremely rare, but devastating, disease of unknown aetiology. We investigated the genetic basis of this autosomal recessive condition in a pair of affected siblings who have 40-year histories of catastrophic gastrointestinal and extraintestinal disease. DESIGN: Genome-wide single-nucleotide polymorphism homozygosity mapping in the two affected family members combined with whole-exome sequencing of one affected sibling. This was followed by confirmatory Sanger sequencing of the likely disease-causing sequence variant and functional studies in affected and unaffected family members. RESULTS: Insertion/deletion variation analysis revealed the presence of a homozygous 4 bp deletion (g.155574_77delGTAA) in the PLA2G4A gene, located in the splice donor site directly after exon 17 (the penultimate exon) of the gene in both affected siblings. This introduces a frameshift of 10 amino acids before a premature stop codon (p.V707fsX10), which is predicted to result in the loss of 43 amino acids (residues 707-749) at the C-terminus of cytosolic phospholipase A2-α (cPLA(2)α). cPLA(2)α protein expression was undetectable in the gut of both siblings, with platelet aggregation and thromboxane A(2) production, as functional assays for cPLA(2)α activity, grossly impaired. CONCLUSIONS: We have identified mutations in PLA2G4A as a cause of CMUSE in two affected siblings. Further studies are needed to determine if mutations in this gene are also responsible for disease of a similar phenotype in other cases.
    Gut 12/2012; · 10.73 Impact Factor
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    ABSTRACT: Prostacyclin is an antithrombotic hormone produced by the endothelium, whose production is dependent on cyclooxygenase (COX) enzymes of which two isoforms exist. It is widely believed that COX-2 drives prostacyclin production and that this explains the cardiovascular toxicity associated with COX-2 inhibition, yet the evidence for this relies on indirect evidence from urinary metabolites. Here we have used a range of experimental approaches to explore which isoform drives the production of prostacyclin in vitro and in vivo. Our data show unequivocally that under physiological conditions it is COX-1 and not COX-2 that drives prostacyclin production in the cardiovascular system, and that urinary metabolites do not reflect prostacyclin production in the systemic circulation. With the idea that COX-2 in endothelium drives prostacyclin production in healthy individuals removed, we must seek new answers to why COX-2 inhibitors increase the risk of cardiovascular events to move forward with drug discovery and to enable more informed prescribing advice.
    Proceedings of the National Academy of Sciences 10/2012; 109(43):17597-602. · 9.81 Impact Factor
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    ABSTRACT: Background H2S is endogenously produced from l-cysteine (L-cys) by the action of three key enzymes cysthationine β-synthase (CBS), cysthationine-γ-lyase (CSE) and the newly discovered 3-mercaptopyruvate sulfurtransferase (3-MST) and . It is present in human blood at micromolar concentrations [3] and it is involved in the maintenance of cardiovascular homeostasis [4]. To date, the influence of H2S on platelets, which play a central role in blood homeostasis, has been poorly explored. Therefore, we aimed to evaluate the effect of H2S signaling in human platelets. Methods Human washed platelets were collected from 15 healthy volunteers. The expression of both CBS, CSE and 3-MST was evaluated by Western blot analysis. The enzymes activity was evaluated through H2S measurement by a colorimetric assay [5]. Light transmission aggregometry technique was used to analyze platelet aggregation. H2S-induced effect was evaluated using both an exogenous source of H2S, sodium hydrogen sulphide (NaHS, 0.1 μM–10 mM) and the metabolic precursor, L-cys (0.1 μM–10 mM) on thrombin receptor activator peptide 6 amide (TRAP-6, 2 μM) stimulus. We operated a pharmacological modulation by using specific inhibitors of arachidonic acid cascade, the main pathway involved in platelet function. Indomethacin (INDO, 10 μM, 15 min), a COX inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3, 1 μM, 15 min), a phospholipase A2 (PLA2) inhibitor or SQ29548 (1 μM), a thromboxane receptor antagonist were used. In addition, thromboxane (TXA2) and cAMP levels were evaluated. Results Human washed platelets expressed CBS, CSE and 3-MST and generated detectable amounts of H2S. Incubation with L-cys significantly increased H2S release (p < 0.001). Neither L-cys nor NaHS (0.1 μM–10 mM) affected human washed platelets in resting conditions, but both significantly increased TRAP-6-induced aggregation (p < 0.001 for L-cys 0.1 mM and NaHS 0.1 mM and 10 μM; p < 0.01 for NaHS 1 μM and p < 0.05 for L-cys 10 μM). Besides, H2S did not modify platelet cAMP levels. Conversely, INDO, AACOCF3 and SQ29548 blocked the potentiating effect of H2S on platelet aggregation induced by TRAP-6. Interestingly, both NaHS and L-cys induced a significantly increase in TXA2production (p < 0.01). Conclusions Our data imply that the H2S endogenously produced within human platelets is involved in platelet aggregation through PLA2 activation. These findings may open a new pharmacological approache in platelet dependent disorders.
    Nitric Oxide 09/2012; 27:S31–S32. · 3.27 Impact Factor
  • Timothy David Warner, Jane Alison Mitchell, Nicholas Sebastian Kirkby
    Heart (British Cardiac Society) 05/2012; 98(9):679-80. · 5.01 Impact Factor
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    ABSTRACT: The potent vasoconstrictor endothelin-1 (ET-1), acting on the endothelin-A (ETA) receptor, promotes intimal lesion formation following vascular injury. The endothelin-B (ETB) receptor, which mediates nitric oxide release and ET-1 clearance in endothelial cells, may moderate lesion formation, but this is less clear. We used selective ET receptor antagonists and cell-specific deletion to address the hypothesis that ETB receptors in the endothelium inhibit lesion formation following arterial injury. Neointimal proliferation was induced by wire or ligation injury to the femoral artery in mice treated with selective ETA (ABT-627) and/or ETB antagonists (A192621). Measurement of lesion formation by optical projection tomography and histology indicated that ETA blockade reduced lesion burden in both models. Although ETB blockade had little effect on ligation injury-induced lesion formation, after wire injury, blockade of the ETB receptor increased lesion burden (184% of vehicle; P < 0.05) and reversed the protective effects of an ETA antagonist. Selective deletion of ETB receptors from the endothelium, however, had no effect on neointimal lesion size. These results are consistent with ETB receptor activation playing an important role in limiting neointimal lesion formation following acute vascular injury, but indicate that this protective effect is not mediated by those ETB receptors expressed by endothelial cells. These data support the proposal that selective ETA antagonists may be preferable to mixed ETA/ETB antagonists for targeting the arterial response to injury.
    Cardiovascular Research 03/2012; 95(1):19-28. · 5.81 Impact Factor
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    ABSTRACT: This study evaluated the effect of prasugrel alone, and in combination with low and high dose aspirin, on urinary metabolites of thromboxane A2 (TXA2) and prostaglandin I2 (PGI2), TX-M and PGI-M, respectively. 9 healthy males, aged 18-40, were enrolled in the 21-day study. Prasugrel was loaded at 60 mg on day 1 and maintained at 10 mg once daily until day 21. At day 8 aspirin 75 mg o.d. was introduced and the dose increased to 300 mg o.d. on day 15. On days 0, 7, 14 and 21 urine samples were obtained and TX-M and PGI-M assayed by mass spectrometry. Platelet aggregation to a TXA2-mimetic (U46619) was also determined at each time point. Data are presented as ng of urinary metabolite per mg creatine (mean±SEM). Data were analysed by one-way ANOVA with Bonferroni's post-test. Prasugrel alone did not reduce either TX-M (day 7, 0.24±0.02 ng per mg creatine; day 0, 0.28±0.04) or PGI-M (day 7, 0.15±0.03; day 0, 0.15±0.04), although both metabolites were reduced by the addition of low dose aspirin (day 14: TX-M, 0.09±0.01; PGI-M, 0.07±0.01; p<0.01 vs day 0). There were no further effects with the higher aspirin dose (p>0.05). Platelet aggregation to U46619 was largely inhibited by prasugrel, with no further effect of aspirin. Prasugrel alone preserves urinary PGI2 metabolites while inhibiting TXA2-driven platelet aggregation. Addition of aspirin reduces the excreted levels of metabolites of both TXA2 and vasoprotective PGI2.
    Heart (British Cardiac Society) 10/2011; 97(20):e7. · 5.01 Impact Factor
  • T Hoefer, N S Kirkby, T D Warner
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    ABSTRACT: Inhibition of platelet aggregation following aspirin ingestion is thought to be overcome by the presence of a small minority of uninhibited platelets. Indeed, it is often quoted that more than 95% of platelets must be inhibited by aspirin for full anti-thrombotic protection.1 2 Aspirin is an irreversible inhibitor of platelet cyclooxygenase, while the thienopyridines, notably clopidogrel and prasugrel, are irreversible blockers of platelet P2Y12 receptors. In analogy to the effects of aspirin, we have investigated the relationships between the proportion of P2Y12 inhibited platelets and aggregation responses, using the agonists ADP, U46619, TRAP-6 amide and collagen. Platelet rich plasma obtained from healthy volunteers was incubated with either prasugrel-active metabolite (PAM; 3 μM) or vehicle. After washing, different combinations of PAM- and vehicle-treated platelets were transferred to 96-well plates containing fibrinogen (10 μM), CaCl(2) (2 μM) and increasing concentrations of ADP, U46619, TRAP-6 amide or collagen. To follow platelet aggregation, absorbance at 595 nm was measured over 16 min of vigorous shaking. In contrast to studies with aspirin, in this in vitro study we have found a linear relationship between the proportion of P2Y12 receptor uninhibited platelets and aggregation in responses to ADP, U46619 and TRAP-6 amide. As P2Y12 receptor antagonists are almost always used in conjunction with aspirin for anti-thrombotic protection this suggests a complex relationship between proportions of uninhibited platelets and platelet responsiveness which demands further examination, particularly with regard to at risk patient groups.
    Heart (British Cardiac Society) 10/2011; 97(20):e7. · 5.01 Impact Factor

Publication Stats

229 Citations
175.98 Total Impact Points

Institutions

  • 2010–2014
    • Imperial College London
      Londinium, England, United Kingdom
  • 2012–2013
    • William Harvey Research Institute
      Londinium, England, United Kingdom
  • 2010–2013
    • Queen Mary, University of London
      • Barts and The London School of Medicine and Dentistry
      Londinium, England, United Kingdom
  • 2008–2013
    • The University of Edinburgh
      • Centre for Cardiovascular Science
      Edinburgh, Scotland, United Kingdom