Nicola Englyst

Aspirin resistance is more common in lacunar strokes than embolic strokes and is related to stroke severity

The aim of this study was to investigate the relationship between aspirin resistance, ischaemic stroke subtype, stroke severity, and inflammatory cytokines. Aspirin resistance was assessed by thrombelastography in 45 people with ischaemic stroke and 25 controls. Plasma interleukin (IL)-6 was measured. Stroke severity was assessed using the modified Rankin scale and National Institute of Health Stroke Score within 72 h of stroke. Aspirin resistance was more common in the stroke than the control group (67% versus 40%, P=0.028), and within the stroke group the aspirin-resistant group had a higher Rankin score (4.0 versus 2.0, P=0.013). Aspirin resistance was greater in lacunar than embolic strokes (platelet activation 79% versus 59%, P=0.020). The stroke aspirin-resistant group had higher levels of IL-6 than the stroke aspirin-sensitive group (2.4±1 versus 1.8±0.9 ng/mL, P=0.037). Using multivariate analysis, we examined the interrelationships between aspirin resistance, IL-6, and stroke severity. These analyses showed that IL-6 was independently associated with stroke severity as the outcome (B=3.738, P=0.036), and aspirin resistance was independently associated with IL-6 (B=0.765, P=0.005) as the outcome. In conclusion, aspirin resistance is related to stroke severity and aspirin resistance is more common in lacunar strokes than embolic strokes.

Effect of clopidogrel withdrawal on platelet reactivity and vascular inflammatory biomarkers 1 year after drug-eluting stent implantation: results of the prospective, single-centre CESSATION study

Background The optimal duration of clopidogrel treatment, particularly following drug-eluting stent (DES) implantation, remains contentious. Previous studies have observed a clustering of adverse events following clopidogrel cessation 1 year after DES, the aetiology of which is poorly understood. Objective To investigate, in the prospective CESSATION study, the effect of clopidogrel withdrawal at 1 year after DES implantation on (i) arachidonic acid (AA)- and adenosine diphosphate (ADP)-induced platelet aggregation, and (ii) biomarkers of vascular inflammation, including soluble CD40 ligand (sCD40L), high-sensitivity C-reactive protein (hsCRP) and interleukin 6 (IL-6). Methods and results The prospective CESSATION study was undertaken in 33 patients receiving aspirin and due to discontinue clopidogrel 1 year after DES. Platetet reactivity was measured using short thromboelastography, and compliance with aspirin determined from serum thromboxane B2 (TXB2) levels. Venesection was performed at 4 weeks and 24 h before, and at 24 h, 48 h, 1, 2 and 4 weeks after, clopidogrel cessation. Following clopidogrel withdrawal, there was (i) a predictable increase in ADP-induced platelet aggregation (ii) an unexpected significant increase in AA-induced platelet aggregation (iii) a decline in IL-6 and hsCRP at 1 week and 4 weeks respectively; and (iv) a non-significant increase in sCD40L at 4 weeks TXB2 levels were consistently suppressed, indicating complete inhibition of cyclo-oxygenase-1 by aspirin. Conclusion An aspirin-independent, time-dependent increase in AA-induced platelet activation following clopidogrel withdrawal in patients with a DES has been described. New insights into a potential mechanism for the observed clustering of adverse events that occur early after clopidogrel cessation have been provided. These findings raise the question as to whether AA-induced clotting is an appropriate test of aspirin sensitivity.

IL-6 is a predictive biomarker for stroke associated infection and future mortality in the elderly after an ischemic stroke

BACKGROUND AND PURPOSE Stroke associated infection (within the first seven days) occurs in approximately half of stroke patients and is associated with a worse prognosis, especially in the elderly. It is uncertain what factors predict stroke associated infection, yet identification of a suitable biomarker for infection may allow early and appropriate intervention with antibiotics. The aims of this study were to: a) identify independent risk factors for stroke associated infection, and b) test relationships between these risk factors and mortality at 2 years. METHODS Eight-two elderly patients were assessed within 72 h of stroke. Data on stroke severity (Barthel Index), stroke associated infection and mortality at 2 years were collected. Inflammatory biomarkers at baseline and 6 months were measured by ELISA. Logistic regression was used to identify risk factors for stroke associated infection and death. RESULTS Patients with stroke associated infection, especially pneumonia, had increased IL-6, more severe strokes, and higher mortality. IL-6 was independently associated with stroke associated infection (OR = 19.2, [95%CI 3.68, 100], p < 0.001), after adjustment for other risk factors and cytokines. IL-6 was also independently associated with 2 year mortality (OR = 9.2, [1.0, 85.1], p = 0.031). CONCLUSIONS These data suggest that IL-6 may be a key biomarker for predicting stroke associated infection and mortality in the first two years post stroke.

Extracellular Vesicle Flow Cytometry Analysis and Standardization

The term extracellular vesicles (EVs) describes membranous vesicles derived from cells, ranging in diameter from 30 to 1,000 nm with the majority thought to be in the region of 100–150 nm. Due to their small diameter and complex and variable composition, conventional techniques have struggled to accurately count and phenotype EVs. Currently, EV characterization using high-resolution flow cytometry is the most promising method when compared to other currently available techniques, due to it being a high-throughput, single particle, multi-parameter analysis technique capable of analyzing a large range of particle diameters. Whilst high resolution flow cytometry promises detection of the full EV diameter range, standardization of light scattering and fluorescence data between different flow cytometers remains an problem. In this mini review, we will discuss the advances in high-resolution flow cytometry development and future direction of EV scatter and fluorescence standardization. Standardization and therefore reproducibility between research groups and instrumentation is lacking, hindering the validation of EVs use as diagnostic biomarkers and therapeutics.

Infection and IL-6 are independent risk factors for a poor functional outcome from ischemic stroke

Background: Infections cause an inflammatory response that may activate the endothelial cells lining blood vessels. Infections affect stroke outcome but the mechanisms have not been elucidated. Our hypothesis is that inflammation due to infections may increase endothelial cell activation, causing prothrombotic changes in coagulation, thereby causing more severe strokes. Methods: People suffering an ischemic stroke in the last 72 hours (n=85) were recruited. Information on infections was collected from patient notes. ELISAs were used to measure plasma markers of endothelial cell activation, inflammation and coagulation. Results: 32% of people with an acute stroke had an infection in the 7 days post stroke. There were no differences in characteristics of the groups, except that 77% of people without an infection took aspirin compared with 46% of people with an infection (p=0.005). Infection, in particular pneumonia, was related to the subclass of stroke (OCSP) (p=0.036, with more TACS and fewer LACS in the infection group). There was a trend for those with an infection to have a larger infarct volume. Stroke functional outcome was worse in those people with an infection (p<0.001 for Rankin’s, Barthel and NIHSS scores). Infection was also related to destination on discharge. People with infections had increased inflammation (IL-6: p<0.001), increased coagulation (F1+2, p=0.048) and a trend for increased endothelial activation (vWF, p=0.125). IL-6 was correlated with vWF (R=0.371, p<0.001) and F1+2 (0.210, p=0.079). 41% of the variation in functional outcome was explained by linear regression models containing infection + IL-6 + stroke subtype, or 28% by infection + IL-6 + infarct volume. Discussion: These data are consistent with our hypothesis that infection increases inflammation, resulting in a poorer functional outcome. We suggest that IL-6 is associated with endothelial cell activation and coagulation, and that infection and IL-6 are independent risk factors for a poor functional outcome.

Platelet Microvesicles (Microparticles) in Cardiac Surgery

SIGNIFICANT POSTOPERATIVE BLEEDING is a common risk of cardiac surgery, with approximately 3.5% of patients requiring surgical re-exploration.1 Re-exploration is associated with adverse outcomes, including infections, ischemia, and increased 30-day mortality.2 Similar adverse outcomes are related to erythrocyte transfusions associated with cardiac surgery,3 in addition to the immunologic and administrative hazards of transfusion.4 These risks are important because the majority of patients undergoing cardiac surgery receive a blood transfusion despite the lack of evidence to support liberal transfusion strategies.5 The frequency and significance of bleeding after cardiac surgery warrant investigation of the hematologic changes throughout the procedure. This review focuses on the (patho)physiology of platelet-derived microvesicles in the setting of cardiovascular surgery, a developing area in the understanding of the control of coagulation.

Changes in platelet function with inflammation in patients undergoing vascular surgery

Abstract The role of platelets in ischaemic events is well established. Aspirin represents the default antiplatelet and blocks the metabolism of arachidonic acid (AA) at the cyclo-oxygenase enzyme (COX). AA is commonly used as a test of response to aspirin, but recent data raise uncertainty about the validity of this approach. Specifically, in some patients AA-induced clotting is not suppressed, but the level of COX-dependent AA metabolite, thromboxane B2 (TXB2) is negligible. Furthermore, AA-induced whole blood clotting varies dynamically in individuals, who are aspirin responsive according to TXB2 levels. The aim of this study was to assess the level of AA-, ADP- and thrombin-mediated platelet reactivity in patients on aspirin before, during, and after major vascular surgery, which represents a model of on/off vascular inflammation. Firstly, we hypothesized, that in association with this inflammatory episode AA-, ADP- and thrombin-induced clotting would change in a dynamic manner. Secondly, that AA-induced clotting will be modified despite complete suppression of platelet TXB2 production by aspirin throughout the periprocedural period, possibly via a lipoxygenase-mediated mechanism. Fourty patients underwent major vascular surgery (open abdominal aortic aneurysm operation, infrainguinal bypass for subcritical limb ischaemia or peripheral aneurysm repair with bypass). They were all on 75 mg of aspirin prior to and throughout the perioperative period and received 5000 units of unfractionated heparin intraoperatively. AA-, ADP-, and thrombin-induced clotting, AA metabolites (TXB2 and 12-Hyroxyeicosatetraenoic acid (12-HETE)) and inflammatory markers (CRP, IL-6, TNF-α and CD40) were measured pre-procedure and at 2, 24, 48 hours, 3 to 5 days and 3 months after surgery. AA-, ADP- and thrombin-induced platelet reactivity was assessed using thrombelastography. TXB2, 12-HETE, IL-6, TNF-α, CD40 were determined using the sequential competitive binding Enzyme-Linked ImmunoAssay technique and CRP was determined using an immune-turbidimetric test on human serum. There was a transient rise in inflammatory markers in the early perioperative period (CRP at 24, 48 hours and 3 to 5 days p < 0.001 and IL-6 at 2, 24, 48 hours and 3 to 5 days p < 0.001 as compared to baseline). Patients had negligible levels of TXB2 throughout, confirming a consistent therapeutic response to aspirin. There was a transient rise in thrombin-mediated clotting (MAThrombin at 48 hours p = 0.001 and 3 to 5 days p < 0.001) and a fall in AA- and ADP-induced clotting in the early post op period (both MAAA and MAADP p = 0.001 at 2 hours). At 3 months, the level of AA- and ADP-induced clotting was significantly higher than at baseline (p = 0.008 for MAAA and p = 0.002 for MAADP), hence demonstrating a rebound effect. These data demonstrate a novel dynamic variation in platelet aggregation with acute vascular inflammation, including AA-induced whole blood clotting which is apparently COX-1 independent.

Does the response to aspirin and clopidogrel vary over 6 months in patients with ischemic heart disease

Dual‐antiplatelet therapy (DAPT) with aspirin and a P2Y12 inhibitor, mostly clopidogrel, is the default therapy in both acute coronary syndrome (ACS) and after intracoronary stents. It is well established that responses to antiplatelet therapy (APT), particularly clopidogrel, are subject to considerable interindividual variability.

Microvesicles as Biomarkers in Diabetes, Obesity and Non-Alcoholic Fatty Liver Disease: Current Knowledge and Future Directions

NAFLD is the most common chronic liver disease, frequently associated with diabetes. Both of these insulin resistant states have increased cardiovascular risk factors associated, and a prevalent cause of mortality in these diseases. Microvesicles are heterogonously sized, phospholipid rich spheres released by cells upon activation and apoptosis. Evidence is continuing to accumulate of microvesicles being not only markers of disease severity but as also having a functional role in the pathophysiology of disease progression.

Leukocyte extracellular vesicle concentration is inversely associated with liver fibrosis severity in NAFLD

The enhanced liver fibrosis (LFS) score and the nonalcoholic fatty liver disease (NAFLD) fibrosis score (NFS) are algorithmic‐derived scores for diagnosing severe (F3/F4) liver fibrosis. In a pilot, substudy of the Wessex Evaluation of fatty Liver and Cardiovascular markers in NAFLD with OMacor thErapy (WELCOME) trial, we tested whether measurements of plasma platelet‐, endothelial‐, and leukocyte‐derived extracellular vesicles (EVs) counts are (a) associated with, and predict, F3/F4 fibrosis and (b) able to improve risk prediction of F3/F4 fibrosis in NAFLD, building upon LFS or NFS algorithms. Twenty‐six individuals with NAFLD had liver fibrosis severity determined by Kleiner scoring after liver biopsy. Plasma samples stained with CD41a, CD42b, CD31, CD105, CD14, CD16, and CD284 antibodies were analyzed using flow cytometry to measure platelet‐, endothelial‐, and leukocyte‐derived EVs counts. The independence of associations between EVs and F3/F4 fibrosis were tested using logistic regression. Receiver operator characteristic (ROC) curves were used to evaluate F3/F4 fibrosis prediction models. LFS was more strongly associated with F3/F4 fibrosis than NFS ( χ2 = 15.403, P < 0.0001, and χ2 = 6.300, P = 0.012, respectively). The association between LFS and F3/F4 fibrosis was further improved by addition of CD14+ EVs ( χ2=20.847,P = 0.016 vs. χ2=12.803,P = 0.015, respectively) or CD16+ EVs ( χ2=22.205,P = 0.009 vs. χ2=17.559,P = 0.001, respectively), and the area under the ROC for LFS (AUC = 0.915, se = 0.055, P = 0.001) was increased by the addition of CD14+ or CD16+ EVs (AUC = 0.948, se = 0.042, and P < 0.001 and AUC = 0.967, se = 0.055, P < 0.001, respectively) as predictor variables. In this small preliminary study, CD14+ and CD16+ EV counts show potential to predict liver fibrosis severity with either marker improving the ability of the LFS to identify F3/F4 fibrosis in this small preliminary cohort study.