Ticagrelor, but not clopidogrel, reduces arterial thrombosis via endothelial tissue factor suppression

The P2Y12 antagonist ticagrelor reduces mortality in patients with acute coronary syndrome (ACS), compared with clopidogrel, and the mechanisms underlying this effect are not clearly understood. Arterial thrombosis is the key event in ACS; however, direct vascular effects of either ticagrelor or clopidogrel with focus on arterial thrombosis and its key trigger tissue factor have not been previously investigated.Methods and results: Human aortic endothelial cells were treated with ticagrelor or clopidogrel active metabolite (CAM) and stimulated with tumour necrosis factor-alpha (TNF-α); effects on procoagulant tissue factor (TF) expression and activity, its counter-player TF pathway inhibitor (TFPI) and the underlying mechanisms were determined. Further, arterial thrombosis by photochemical injury of the common carotid artery, and TF expression in the murine endothelium were examined in C57BL/6 mice treated with ticagrelor or clopidogrel. Ticagrelor, but not CAM, reduced TNF-α-induced TF expression via proteasomal degradation and TF activity, independently of the P2Y12 receptor and the equilibrative nucleoside transporter 1 (ENT1), an additional target of ticagrelor. In C57BL/6 mice, ticagrelor prolonged time to arterial occlusion, compared with clopidogrel, despite comparable antiplatelet effects. In line with our in vitro results, ticagrelor, but not clopidogrel, reduced TF expression in the endothelium of murine arteries.Conclusion: Ticagrelor, unlike clopidogrel, exhibits endothelial-specific antithrombotic properties and blunts arterial thrombus formation. The additional antithrombotic properties displayed by ticagrelor may explain its greater efficacy in reducing thrombotic events in clinical trials. These findings may provide the basis for new indications for ticagrelor

Interleukin-1\u3b2 Mediates Arterial Thrombus Formation via NET-Associated Tissue Factor

CANTOS reported reduced secondary atherothrombotic events in patients with residual inflammatory risk treated with the inhibitory anti-IL-1\u3b2 antibody, Canakinumab. Yet, mechanisms that underlie this benefit remain elusive. Recent work has implicated formation of neutrophil extracellular traps (NETosis) in arterial thrombosis. Hence, the present study explored the potential link between IL-1\u3b2, NETs, and tissue factor (TF)-the key trigger of the coagulation cascade-in atherothrombosis. To this end, ST-elevation myocardial infarction (STEMI) patients from the Swiss multicenter trial SPUM-ACS were retrospectively and randomly selected based on their CRP levels. In particular, 33 patients with STEMI and high C-reactive protein (CRP) levels ( 65 10 mg/L) and, 33 with STEMI and low CRP levels ( 64 4 mg/L) were investigated. High CRP patients displayed elevated circulating IL-1\u3b2, NETosis, and NET-associated TF plasma levels compared with low CRP ones. Additionally, analysis of patients stratified by circulating IL-1\u3b2 levels yielded similar results. Moreover, NETosis and NET-associated TF plasma levels correlated positively in the whole population. In addition to the above, translational research experiments provided mechanistic confirmation for the clinical data identifying IL-1\u3b2 as the initial trigger for the release of the pro-coagulant, NET-associated TF. In conclusion, blunted TF presentation by activated neutrophils undergoing NETosis may provide a mechanistic explanation to reduced secondary atherothrombotic events as observed in canakinumab-treated patients in CANTOS

Omega-3 Fatty Acids and Heart Rhythm, Rate, and Variability in Atrial Fibrillation.

Background Previous randomized control trials showed mixed results concerning the effect of omega-3 fatty acids (n-3 FAs) on atrial fibrillation (AF). The associations of n-3 FA blood levels with heart rhythm in patients with established AF are unknown. The goal of this study was to assess the associations of total and individual n-3 FA blood levels with AF type (paroxysmal versus nonparoxysmal), heart rate (HR), and HR variability in patients with AF. Methods and Results Total n-3 FAs, eicosapentaenoic acid, docosahexaenoic acid, docosapentaenoic acid, and alpha-linolenic acid blood levels were determined in 1969 patients with known AF from the SWISS-AF (Swiss Atrial Fibrillation cohort). Individual and total n-3 FAs were correlated with type of AF, HR, and HR variability using standard logistic and linear regression, adjusted for potential confounders. Only a mild association with nonparoxysmal AF was found with total n-3 FA (odds ratio [OR], 0.97 [95% CI, 0.89-1.05]) and docosahexaenoic acid (OR, 0.93 [95% CI, 0.82-1.06]), whereas other individual n-3 FAs showed no association with nonparoxysmal AF. Higher total n-3 FAs (estimate 0.99 [95% CI, 0.98-1.00]) and higher docosahexaenoic acid (0.99 [95% CI, 0.97-1.00]) tended to be associated with slower HR in multivariate analysis. Docosapentaenoic acid was associated with a lower HR variability triangular index (0.94 [95% CI, 0.89-0.99]). Conclusions We found no strong evidence for an association of n-3 FA blood levels with AF type, but higher total n-3 FA levels and docosahexaenoic acid might correlate with lower HR, and docosapentaenoic acid with a lower HR variability triangular index

Endothelial SIRT6 deficiency promotes arterial thrombosis in mice

OBJECTIVE Arterial thrombosis may be initiated by endothelial inflammation or denudation, activation of blood-borne elements or the coagulation system. Tissue factor (TF), a central trigger of the coagulation cascade, is regulated by the pro-inflammatory NF-κB-dependent pathways. Sirtuin 6 (SIRT6) is a nuclear member of the sirtuin family of NAD$^{+}$-dependent deacetylases and is known to inhibit NF-κB signaling. Its constitutive deletion in mice shows early lethality with hypoglycemia and accelerated aging. Of note, the role of SIRT6 in arterial thrombosis remains unknown. Thus, we hypothesized that endothelial SIRT6 protects from arterial thrombosis by modulating inhibition of NF-κB-associated pathways. APPROACH AND RESULTS Using a laser-induced carotid thrombosis model, in vivo arterial occlusion occurred 45% faster in 12-week-old male endothelial-specific Sirt6$^{-/-}$ mice as compared to Sirt6$^{fl/fl}$ controls (n ≥ 9 per group; p = 0.0012). Levels of procoagulant TF were increased in animals lacking endothelial SIRT6 as compared to control littermates. Similarly, in cultured human aortic endothelial cells, SIRT6 knockdown increased TF mRNA, protein and activity. Moreover, SIRT6 knockdown increased mRNA levels of NF-κB-associated genes tumor necrosis factor alpha (TNF-α), poly [ADP-ribose] polymerase 1 (PARP-1), vascular cell adhesion molecule 1 (VCAM-1), and cyclooxygenase-2 (COX-2); at the protein level, COX-2, VCAM-1, TNF-α, and cleaved PARP-1 remained increased after Sirt6 knockdown. CONCLUSIONS Endothelium-specific Sirt6 deletion promotes arterial thrombosis in mice. In cultured human aortic endothelial cells, SIRT6 silencing enhances TF expression and activates pro-inflammatory pathways including TNF-α, cleaved PARP-1, VCAM-1 and COX-2. Hence, endogenous endothelial SIRT6 exerts a protective role in experimental arterial thrombosis

A theory-based process evaluation alongside a randomised controlled trial of printed educational messages to increase primary care physicians' prescription of thiazide diuretics for hypertension [ISRCTN72772651]

Background Pragmatic trials of implementation interventions focus on evaluating whether an intervention changes professional behaviour under real-world conditions rather than investigating the mechanism through which change occurs. Theory-based process evaluations conducted alongside pragmatic randomised trials address this by assessing whether the intervention changes theoretical constructs proposed to mediate change. The Ontario Printed Educational Materials (PEM) cluster trial was designed to increase family physicians’ guideline-recommended prescription of thiazide diuretics. The trial found no intervention effect. Using the theory of planned behaviour (TPB), we hypothesised that changes in thiazide prescribing would be reflected in changes in intention, consistent with changes in attitude and subjective norm, with no change to their perceived behavioural control (PBC), and tested this alongside the RCT. Methods We developed and sent TPB postal questionnaires to a random sub-sample of family physicians in each trial arm 2 months before and 6 months after dissemination of the PEMs. We used analysis of covariance to test for group differences using a 2 × 3 factorial design. We content-analysed an open-ended question about perceived barriers to thiazide prescription. Using control group data, we tested whether baseline measures of TPB constructs predicted self-reported thiazide prescribing at follow-up. Results Four hundred twenty-six physicians completed pre- and post-intervention questionnaires. Baseline scores on measures of TPB constructs were high: intention mean = 5.9 out of 7 (SD = 1.4), attitude mean = 5.8 (SD = 1.1), subjective norm mean = 5.8 (SD = 1.1) and PBC mean = 6.2 (SD = 1.0). The arms did not significantly differ post-intervention on any of the theory-based constructs, suggesting a possible ceiling effect. Content analysis of perceived barriers suggested post-intentional barriers to prescribing thiazides most often focused on specific patient clinical characteristics and potential side effects. Baseline intention (β = 0.63, p < 0.01) but not PBC (β = 0.04, p = 0.78) predicted 42.6 % of the variance in self-reported behaviour at follow-up in the control group. Conclusions Congruent with the Ontario Printed Educational Messages trial results and aligned with the TPB, we saw no impact of the intervention on any TPB constructs. The theoretical basis of this evaluation suggests possible explanations for the failure of the PEM intervention to change professional behaviour, which can directly inform the design and content of future theory-based PEM interventions to change professional behaviour

Glycoprotein Ib clustering in platelets can be inhibited by alpha-linolenic acid as revealed by cryo-electron tomography

Platelet adhesion to the sub-endothelial matrix and damaged endothelium occurs through a multi-step process mediated in the initial phase by glycoprotein Ib binding to von Willebrand factor, which leads to the subsequent formation of a platelet plug. The plant-derived omega-3 fatty acid alpha-linolenic acid is an abundant alternative to fish-derived n-3 FA and has anti-inflammatory and anti-thrombotic properties. In this study, we investigated the impact of alpha-linolenic acid on human platelet binding to vWF under high-shear flow conditions (mimicking blood flow in stenosed arteries). Pre-incubation of fresh human blood from healthy donors with alpha-linolenic acid at dietary relevant concentrations reduced platelet binding and rolling on vWF-coated microchannels at a shear rate of 100 dyn/cm2. Depletion of membrane cholesterol by incubation of platelet-rich-plasma with methyl-beta cyclodextrin abrogated platelet rolling on vWF. Analysis of glycoprotein Ib by applying cryo-electron tomography to intact platelets revealed local clusters of glycoprotein Ib complexes, upon shear-force exposure, whose formation could be prevented by alpha-linolenic acid treatment. This study provides novel findings on the rapid local rearrangement of the glycoprotein Ib complexes in response to high-shear flow and highlights the mechanism of in vitro inhibition of platelet binding to and rolling on vWF by alpha-linolenic acid

Interleukin-1β Mediates Arterial Thrombus Formation via NET-Associated Tissue Factor

CANTOS reported reduced secondary atherothrombotic events in patients with residual inflammatory risk treated with the inhibitory anti-IL-1β antibody, Canakinumab. Yet, mechanisms that underlie this benefit remain elusive. Recent work has implicated formation of neutrophil extracellular traps (NETosis) in arterial thrombosis. Hence, the present study explored the potential link between IL-1β, NETs, and tissue factor (TF)-the key trigger of the coagulation cascade-in atherothrombosis. To this end, ST-elevation myocardial infarction (STEMI) patients from the Swiss multicenter trial SPUM-ACS were retrospectively and randomly selected based on their CRP levels. In particular, 33 patients with STEMI and high C-reactive protein (CRP) levels (≥ 10 mg/L) and, 33 with STEMI and low CRP levels (≤ 4 mg/L) were investigated. High CRP patients displayed elevated circulating IL-1β, NETosis, and NET-associated TF plasma levels compared with low CRP ones. Additionally, analysis of patients stratified by circulating IL-1β levels yielded similar results. Moreover, NETosis and NET-associated TF plasma levels correlated positively in the whole population. In addition to the above, translational research experiments provided mechanistic confirmation for the clinical data identifying IL-1β as the initial trigger for the release of the pro-coagulant, NET-associated TF. In conclusion, blunted TF presentation by activated neutrophils undergoing NETosis may provide a mechanistic explanation to reduced secondary atherothrombotic events as observed in canakinumab-treated patients in CANTOS

Long-term dietary supplementation with plant-derived omega-3 fatty acid improves outcome in experimental ischemic stroke

BACKGROUND AND AIMS Early revascularization -the gold standard therapy for ischemic stroke- is often withheld in the elderly population due to high risk of complications. Thus, safe and effective preventive and therapeutic options are needed. The plant-derived omega-3-fatty-acid alpha-linolenic-acid (ALA) has emerged as a novel cardiovascular-protective agent. As of yet, little is known about its potential therapeutic effects on stroke. We hereby aimed to investigate the impact of a clinically relevant long-term dietary intervention with ALA on stroke outcome. METHODS Six month-old C57BL/6 wildtype males were either fed an ALA-rich (high ALA) or a control diet (low ALA) for 12 months. At 18 months, brain ischemia/reperfusion was induced by transient middle cerebral artery occlusion (tMCAO). Stroke size and neurological function were assessed. Functional blood-brain-barrier-(BBB) permeability and protein expression were assessed by immunohistochemistry. Baseline inflammatory markers were measured at 18 months. RESULTS High ALA-fed animals displayed decreased circulating TNF-α levels and Neutrophil-to-Lymphocyte Ratios at 18 months. Stroke size and neurological dysfunction were significantly reduced in high ALA-fed animals. Coherently to the reduced stroke size, functional BBB integrity and occludin endothelial expression were maintained by high ALA supplementation. Additionally, ALA reduced endothelial activation and thus recruitment and activation of macrophages and resident microglia. Finally, high ALA diet reduced the expression of BBB-degrading and neurotoxic MMP-3 and MMP-9. CONCLUSIONS We demonstrate the beneficial effects of a clinically relevant and feasible dietary intervention with a safe and readily available compound in the setting of stroke. The protective effects observed with ALA supplementation may relate to blunting of inflammation and might pave the way for novel stroke treatments

Post-ischaemic administration of the murine Canakinumab-surrogate antibody improves outcome in experimental stroke

Aims The CANTOS trial underscored the efficacy of selective antibody-based interleukin (IL)-1β inhibition with Canakinumab in secondary prevention of cardiovascular events. Despite the success of the trial, incidence of stroke was not reduced likely due to the low number of events and the relatively young age of patients enrolled. Given the established role of IL-1β in stroke, we tested the efficacy of the murine Canakinumab-equivalent antibody in a mouse model of ischaemic stroke. To mimic the clinical scenario of modern stroke management, IL-1β inhibition was performed post-ischaemically upon reperfusion as it would be the case in patients presenting to the emergency room and eligible for thrombolytic therapy. Methods and results Transient middle cerebral artery occlusion (tMCAO) was performed in wild type mice; upon reperfusion, mice were randomly allocated to anti-IL-1β antibody or vehicle treatment. Following tMCAO, cerebral IL-1β levels, unlike tumour necrosis factor-α, were increased underscoring a role for this cytokine. Post-ischaemic treatment with IL-1β antibody reduced infarct size, cerebral oedema and improved neurological performance as assessed by 2,3,5-triphenyltetrazolium chloride staining, Bederson and RotaRod tests. Antibody-treated animals also exhibited a reduced neutrophil and matrix metalloproteinase (MMP)-2 but not MMP-9, activity in ipsilateral hemispheres as compared to vehicle-treated mice. Noteworthy, tMCAO associated vascular endothelial-cadherin reduction was blunted in IL-1β antibody-treated mice compared to vehicle-treated, likely providing the mechanistic explanation for the improved outcome. Conclusion Our data for the first time demonstrate the efficacy of selective post-ischaemic IL-1β blockade in improving outcome following experimental ischaemia/reperfusion brain injury in the mouse and encourage further focused clinical studies assessing the potential of the approved IL-1β antibody Canakinumab, as an adjuvant therapy to thrombolysis in acute ischaemic stroke patients