Melissa V. Chan

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.

HYDROGEN SULFIDE-BASED THERAPEUTICS AND GASTROINTESTINAL DISEASES: TRANSLATING PHYSIOLOGY TO TREATMENTS

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.

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.

Association of MicroRNAs and YRNAs With Platelet Function

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 (rp=0.28; n=121; P=0.002), miR-126 (rp=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 P2Y12 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.

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.

Systematic study of constitutive cyclooxygenase-2 expression: Role of NF-κB and NFAT transcriptional pathways.

Significance Nonsteroidal antiinflammatory drugs (NSAIDs) work by inhibiting cyclooxygenase-2 (COX-2) induced at sites of inflammation. They are among the most widely used drugs worldwide, but their cardiovascular side effects are a major concern for patients, regulators, and industry. NSAID side effects are mediated by inhibition of constitutively expressed COX-2 present in discrete regions, including the kidney. However, the pathways driving constitutive COX-2 remain poorly understood. The work presented here defines these pathways and importantly shows constitutive COX-2 expression in the kidney occurs through pathways distinct to those driving COX-2 in inflammation. These data therefore highlight the potential that targeting COX-2 at the transcriptional level may provide a way to dissociate antiinflammatory benefits of NSAIDs from their treatment-limiting cardiovascular side effects. 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.

Optical multichannel (optimul) platelet aggregometry in 96-well plates as an additional method of platelet reactivity testing.

Platelet reactivity testing is important for the diagnosis of bleeding disorders, and increasingly to optimise anti-platelet therapy. Traditional light transmission aggregometry is considered the gold standard, whilst 96-well plate aggregometry, founded on similar principles, provides a higher throughput screening method. Despite the widespread use of both, methodologies and outputs vary widely between laboratories. We report a methodological approach towards providing a standardised optical detection of platelet aggregation (optimul method) based upon 96-well plate aggregometry. Individual wells of half-area 96-well plates were coated with gelatine and one of seven concentrations of arachidonic acid (AA), adenosine diphosphate (ADP), collagen, epinephrine (EPI), ristocetin, TRAP-6 amide or U46619, before being lyophilised, vacuum-sealed, foil-packed and stored at room temperature for up to 24 weeks. For platelet testing, 40 µl of platelet-rich plasma was added to each well. Platelet aggregation was determined by changes in light absorbance, release of ATP/ADP by luminescence and release of thromboxane (TX) A2 by ELISA. Some experiments were conducted in the presence of aspirin (30 µM) or prasugrel active metabolite (PAM; 3 µM). Optimul plates stored for up to 12 weeks permitted reliable detection of concentration-dependent platelet aggregation, ATP/ADP release and TXA2 production. PAM caused reductions in platelet responses to AA, ADP, collagen, EPI, TRAP-6 and U46619, whilst aspirin inhibited responses to AA, collagen and EPI. We conclude that the optimul method offers a viable, standardised approach, allowing platelet reactivity testing and could provide a broad platelet function analysis without the need for dedicated equipment.

Characterization of multiple platelet activation pathways in patients with bleeding as a high-throughput screening option: use of 96-well Optimul assay

Up to 1% of the population have mild bleeding disorders, but these remain poorly characterized, particularly with regard to the roles of platelets. We have compared the usefulness of Optimul, a 96-well plate-based assay of 7 distinct pathways of platelet activation to characterize inherited platelet defects in comparison with light transmission aggregometry (LTA). Using Optimul and LTA, concentration-response curves were generated for arachidonic acid, ADP, collagen, epinephrine, Thrombin receptor activating-peptide, U46619, and ristocetin in samples from (1) healthy volunteers (n = 50), (2) healthy volunteers treated with antiplatelet agents in vitro (n = 10), and (3) patients with bleeding of unknown origin (n = 65). The assays gave concordant results in 82% of cases (κ = 0.62, P < .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 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 trial was registered at www.isrctn.org as #ISRCTN 77951167.

Distinct endothelial pathways underlie sexual dimorphism in vascular auto-regulation

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.

Standardised optical multichannel (optimul) platelet aggregometry using high-speed shaking and fixed time point readings

To the editor 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 testing could be used alongside light transmission aggregometry (LTA) to provide a broad analysis of platelet function. LTA is considered the ‘gold standard’ for platelet function testing [2]. Unfortunately, only a few samples may be analysed at one time, leading to the requirement of a large volume of blood and local rules such that generally only a small number of different agonists are tested in a limited range of concentrations. A broader range of agonists may be tested using 96-well plate aggregometry [3]. This method, however, is also limited by the need to freshly prepare agonists, which as for LTA limits the assay to specialised laboratories and makes it largely unsuitable for high-throughput testing. Our optimul method has simplified standard 96-well plate aggregometry, with the introduction of lyophilised agonists and the use of a particular plate reader with kinetic shaking over 16 min to limit the inherent variability between 96-well plate methods. We have shown it to provide a standardised ‘off-the-shelf ’ assay, which requires only the addition of 40 ml platelet-rich plasma (PRP) to each agonist-coated well to run the test [1]. Here, we demonstrate that the optimul plates used for the Tecan Sunrise kinetic plate reader may also be used in conjunction with a high-speed thermoshaker. This method further simplifies the assay by removing the requirement for a particular kinetic plate reader and reduces the time required to perform this test. Together, these adaptations increase the overall efficiency and potential costeffectiveness of the test. Optimul plates were prepared as previously described [1]. Briefly, platelet agonists, arachidonic acid (AA), adenosine diphosphate (ADP), collagen, epinephrine (EPI), ristocetin, TRAP-6 amide and U46619 were added to individual wells of a gelatinecoated half-area 96-well plate and lyophilised. These plates were stored vacuum-sealed and light-tight at room temperature until use. Human blood was collected by venepuncture into tri-sodium citrate (3.2% w/v; 1:9 dilution) from healthy volunteers (n1⁄4 4–6) who had abstained from NSAIDs or aspirin for 2 weeks previously. PRP was obtained by centrifugation of whole blood (175 g, 15 min, 25 C) and a platelet count was performed to confirm normal platelet numbers (2–4 10 platelets/ml). Platelet-poor plasma (PPP) was obtained by further centrifugation of PRP (15 000 g, 2 min, 25 C). PRP was incubated for 30 min at 37 C with aspirin (30mM) and/or the P2Y12 receptor antagonist, prasugrel active metabolite (R-138727, PAM; 3mM), or vehicle (final concentrations 0.03% DMSO or ethanol). PRP or PPP (40 ml) was added into the appropriate wells of the optimul plate, the plate was sealed with film and then placed on a high-speed