Nicholas S. Kirkby

Circulating MicroRNAs as Novel Biomarkers for Platelet Activation

Rationale: MicroRNA (miRNA) biomarkers are attracting considerable interest. Effects of medication, however, have not been investigated thus far. Objective: To analyze changes in plasma miRNAs in response to antiplatelet therapy. Methods and Results: Profiling for 377 miRNAs was performed in platelets, platelet microparticles, platelet-rich plasma, platelet-poor plasma, and serum. Platelet-rich plasma showed markedly higher levels of miRNAs than serum and platelet-poor plasma. Few abundant platelet miRNAs, such as miR-24, miR-197, miR-191, and miR-223, were also increased in serum compared with platelet-poor plasma. In contrast, antiplatelet therapy significantly reduced miRNA levels. Using custom-made quantitative real-time polymerase chain reaction plates, 92 miRNAs were assessed in a dose-escalation study in healthy volunteers at 4 different time points: at baseline without therapy, at 1 week with 10 mg prasugrel, at 2 weeks with 10 mg prasugrel plus 75 mg aspirin, and at 3 weeks with 10 mg prasugrel plus 300 mg aspirin. Findings in healthy volunteers were confirmed by individual TaqMan quantitative real-time polymerase chain reaction 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 miRNAs, such as miR-223, miR-191, and others, that is, miR-126 and miR-150, decreased on further platelet inhibition. Conclusions: Our study demonstrated a substantial platelet contribution to the circulating miRNA pool and identified miRNAs responsive to antiplatelet therapy. It also highlights that antiplatelet therapy and preparation of blood samples could be confounding factors in case-control studies relating plasma miRNAs to cardiovascular disease.

The endothelin system as a therapeutic target in cardiovascular disease: great expectations or bleak house?

There is considerable evidence that the potent vasoconstrictor endothelin‐1 (ET‐1) contributes to the pathogenesis of a variety of cardiovascular diseases. As such, pharmacological manipulation of the ET system might represent a promising therapeutic goal. Many clinical trials have assessed the potential of ET receptor antagonists in cardiovascular disease, the most positive of which have resulted in the licensing of the mixed ET receptor antagonist bosentan, and the selective ETA receptor antagonists, sitaxsentan and ambrisentan, for the treatment of pulmonary arterial hypertension (PAH). In contrast, despite encouraging data from in vitro and animal studies, outcomes in human heart failure have been disappointing, perhaps illustrating the risk of extrapolating preclinical work to man. Many further potential applications of these compounds, including resistant hypertension, chronic kidney disease, connective tissue disease and sub‐arachnoid haemorrhage are currently being investigated in the clinic. Furthermore, experience from previous studies should enable improved trial design and scope remains for development of improved compounds and alternative therapeutic strategies. Although ET‐converting enzyme inhibitors may represent one such alternative, there have been relatively few suitable compounds developed, and consequently, clinical experience with these agents remains extremely limited. Recent advances, together with an increased understanding of the biology of the ET system provided by improved experimental tools (including cell‐specific transgenic deletion of ET receptors), should allow further targeting of clinical trials to diseases in which ET is involved and allow the therapeutic potential for targeting the ET system in cardiovascular disease to be fully realized.

The endothelin system as a therapeutic target in cardiovascular disease

There is considerable evidence that the potent vasoconstrictor endothelin‐1 (ET‐1) contributes to the pathogenesis of a variety of cardiovascular diseases. As such, pharmacological manipulation of the ET system might represent a promising therapeutic goal. Many clinical trials have assessed the potential of ET receptor antagonists in cardiovascular disease, the most positive of which have resulted in the licensing of the mixed ET receptor antagonist bosentan, and the selective ETA receptor antagonists, sitaxsentan and ambrisentan, for the treatment of pulmonary arterial hypertension (PAH). In contrast, despite encouraging data from in vitro and animal studies, outcomes in human heart failure have been disappointing, perhaps illustrating the risk of extrapolating preclinical work to man. Many further potential applications of these compounds, including resistant hypertension, chronic kidney disease, connective tissue disease and sub‐arachnoid haemorrhage are currently being investigated in the clinic. Furthermore, experience from previous studies should enable improved trial design and scope remains for development of improved compounds and alternative therapeutic strategies. Although ET‐converting enzyme inhibitors may represent one such alternative, there have been relatively few suitable compounds developed, and consequently, clinical experience with these agents remains extremely limited. Recent advances, together with an increased understanding of the biology of the ET system provided by improved experimental tools (including cell‐specific transgenic deletion of ET receptors), should allow further targeting of clinical trials to diseases in which ET is involved and allow the therapeutic potential for targeting the ET system in cardiovascular disease to be fully realized.

Cyclooxygenase-1, not cyclooxygenase-2, is responsible for physiological production of prostacyclin in the cardiovascular system

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.

IN THE PRESENCE OF STRONG P2Y12 RECEPTOR BLOCKADE, ASPIRIN PROVIDES LITTLE ADDITIONAL INHIBITION OF PLATELET AGGREGATION

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.

Evidence for the Involvement of Type I Interferon in Pulmonary Arterial Hypertension

Rationale: Evidence is increasing of a link between interferon (IFN) and pulmonary arterial hypertension (PAH). Conditions with chronically elevated endogenous IFNs such as systemic sclerosis are strongly associated with PAH. Furthermore, therapeutic use of type I IFN is associated with PAH. This was recognized at the 2013 World Symposium on Pulmonary Hypertension where the urgent need for research into this was highlighted. Objective: To explore the role of type I IFN in PAH. Methods and Results: Cells were cultured using standard approaches. Cytokines were measured by ELISA. Gene and protein expression were measured using reverse transcriptase polymerase chain reaction, Western blotting, and immunohistochemistry. The role of type I IFN in PAH in vivo was determined using type I IFN receptor knockout (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 patients with systemic sclerosis associated PAH. Serum interferon &ggr; inducible protein 10 (IP10; CXCL10) and endothelin 1 were raised and strongly correlated together. IP10 correlated positively with pulmonary hemodynamics 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 remodeling, and raised serum endothelin 1 levels. Conclusions: These data indicate that type I IFN, via an action of IFNAR1, mediates PAH.

Antiplatelet effects of aspirin vary with level of P2Y12 receptor blockade supplied by either ticagrelor or prasugrel

‘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.

Evidence That Links Loss of Cyclooxygenase-2 With Increased Asymmetric Dimethylarginine Novel Explanation of Cardiovascular Side Effects Associated With Anti-Inflammatory Drugs

Background— Cardiovascular side effects associated with cyclooxygenase-2 inhibitor drugs dominate clinical concern. Cyclooxygenase-2 is expressed in the renal medulla where inhibition causes fluid retention and increased blood pressure. However, the mechanisms linking cyclooxygenase-2 inhibition and cardiovascular events are unknown and no biomarkers have been identified. Methods and Results— Transcriptome analysis of wild-type and cyclooxygenase-2−/− mouse tissues revealed 1 gene altered in the heart and aorta, but >1000 genes altered in the renal medulla, including those regulating the endogenous nitric oxide synthase inhibitors asymmetrical dimethylarginine (ADMA) and monomethyl-L-arginine. Cyclo-oxygenase-2−/− mice had increased plasma levels of ADMA and monomethyl-L-arginine and reduced endothelial nitric oxide responses. These genes and methylarginines were not similarly altered in mice lacking prostacyclin receptors. Wild-type mice or human volunteers taking cyclooxygenase-2 inhibitors also showed increased plasma ADMA. Endothelial nitric oxide 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 cyclooxygenase-2 inhibition and systemic vascular dysfunction with nonsteroidal anti-inflammatory drug usage. Conclusions— We identify the endogenous endothelial nitric oxide synthase inhibitor ADMA as a biomarker and mechanistic bridge between renal cyclooxygenase-2 inhibition and systemic vascular dysfunction.

Cryptogenic multifocal ulcerating stenosing enteritis associated with homozygous deletion mutations in cytosolic phospholipase A2-α

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 (cPLA2α). cPLA2α protein expression was undetectable in the gut of both siblings, with platelet aggregation and thromboxane A2 production, as functional assays for cPLA2α 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.

Blockade of the purinergic P2Y12 receptor greatly increases the platelet inhibitory actions of nitric oxide

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.