Computational fluid dynamics (CFD) offers a powerful tool in characterizing the complex biophysical environment inducing by dynamic storage conditions, providing insights often beyond the reach of conventional experimental approaches. As our understanding of platelet (PLT) biology has advanced, increased attention has been directed toward mechanical stresses, attributing shear forces encountered during collection, processing, and storage to an acceleration decline in PLT concentrate (PC) quality. CFD simulations using the volume of fluid model were used to simulate PC storage under varying agitation frequencies. Key parameters assessed include fluid velocity, wall shear stress (WSS), and gas-liquid mass transfer. Agitation increased fluid velocity and WSS while preserving the temporal symmetry characteristic of sinusoidal motion. Enhanced oxygen transfer was observed in open-top containers; however, when accounting for the gas permeability of storage materials, oxygen availability was ultimately constrained by container permeability rather than fluid motion. These results highlight the dual role of agitation: promoting oxygen transfer while simultaneously introducing mechanical stress that may contribute to PLT storage lesions. Importantly, since oxygen supply is limited by container permeability, reducing agitation could minimize shear-induced PLT damage without compromising oxygenation. Future optimization strategies may involve modifying storage container geometry or permeability to further improve oxygen delivery during storage.

Background: Transient ischaemic attack (TIA) confers a high risk of subsequent stroke. Population-level evidence describing how stroke risk after TIA evolves over time is needed to inform effective secondary prevention. Methods: We conducted a national population-based retrospective cohort study of adults with a recorded TIA event in Wales. Time to first subsequent stroke was modelled using multivariable Cox proportional hazards regression and follow-up was split into prespecified intervals (0–7, 8–30, 31–365, and >365 days) to estimate interval-specific associations. Sensitivity analyses were conducted using a generalised linear mixed-effects model (GLMER). Findings: Among 40,957 individuals with a recorded TIA event, 10,572 (25.8%) had a subsequent stroke. Stroke risk since TIA varied over time. Increasing age was associated with progressively higher stroke risk, particularly beyond one year (75–84 years vs 18–44 years: HR 2.25, 95% CI 1.80–2.81). Diabetes was associated with persistently elevated stroke risk across all follow-up periods (0–7 days: HR 1.29, 95% CI 1.14–1.46; >365 days: HR 1.27, 95% CI 1.15–1.41). Elevated cardiovascular risk (QRISK2 ≥10) was associated with increased stroke risk in the early post-TIA period (HR 2.20, 95% CI 1.30–3.72), while unavailable QRISK2 scores were associated with higher risk, beyond one year (HR 4.04, 95% CI 2.42–6.74). Socioeconomic deprivation was associated with gradient increase in longer-term stroke risk especially among the most deprived. Standard coagulation parameters were recorded in only 16.2% of patients. In sensitivity analyses, individuals without any recorded coagulation-related testing had higher odds of subsequent stroke (aOR 1.48, 95% CI 1.38–1.59). Interpretation: Stroke risk following TIA is dynamic, with key predictors showing important time-varying associations. These findings support time-aware risk stratification, enhanced post-TIA monitoring, and the need for and sustained post-TIA care in longer periods.

Fluid viscosity is important in both physical and biological systems, as it helps describe a fluid’s material properties. Recent findings show that the shear stress experienced during storage of blood products can result in damage of platelets making the viscosity of stored platelet concentrates (PC) an important area of study. In this work we characterised the contribution of different components of the PC to viscosity using nanoparticle tracking microrheometry and co-stream microfluidics, to determine whether reduced viscosity approaches can improve PC quality over storage. Validation experiments showed both methods could accurately report on the viscosity of samples, with the microfluidics approach deemed preferable due to the possibility of changing flow rates and therefore studying a range of shear stress. PC quality was assessed over 8 days by measuring platelet count, mean platelet volume (MPV), platelet distribution width (PDW), pH, aggregation, and platelet activation markers. Results showed that PC viscosity depends on the residual plasma following processing and is independent of the stored platelet concentration. Reducing plasma to 10

Stroke is the second leading cause of death worldwide, with both the scale and impact growing annually. Over 25% of ischaemic stroke sufferers have previously had a transient ischaemic attack (TIA), allowing a unique window of opportunity to diagnose, treat and adapt modifiable cardiovascular risk factors. Despite this, clinical teams are limited by a lack of appropriate methodologies to identify TIA patients at greatest risk of stroke, outside of identifying common cardiovascular risk factors. PREDICT-EV adopts a two-pronged approach, looking both prospectively and retrospectively at novel characteristics in TIA patients who go on to suffer with a stroke. A prospective nested, case-controlled design will allow analysis of novel extracellular vesicles, coagulative risk and a host of informative cohort characteristics. Patients and controls will be followed over the duration of the study, with stroke as the primary clinical outcome. Retrospectively, the Secured Anonymised Information Linkage databank, UK, will provide population data to track coagulative risk in TIA patients who do and do not go on to suffer with a stroke over a 20-year period. We anticipate the identification of a suite of novel biomarkers for TIA patients at greatest risk of suffering a stroke to be vastly impactful on stroke prevention strategies.

BACKGROUND AND OBJECTIVES:Platelet transfusions are essential for mitigating the bleeding risk of neonatal patients with thrombocytopenia. As neonatal patients have a small blood volume, adult therapeutic dose platelet units are split into reduced-volume storage containers to maximize the use of the donated product and reduce donor exposure. The shelf-life of platelets stored in reduced-volume containers, however, is limited to 5 days. Agitation in platelet concentrate (PC) storage is thought to promote gaseous exchange by maintaining a gradient of O2 across the bag film; however, recent studies have shown that agitation-induced shear promotes the progressive decline of platelet quality over storage. MATERIALS AND METHODS:Electron paramagnetic resonance oximetry and Fickian diffusion modelling of O2 were used to investigate the differences in O2 availability, by assessing the O2 concentration, oxygen consumption rate (OCR), influx of O2, total PC OCR and O2 distribution in PCs stored under routine conditions in neonatal (Macopharma, VQE605B) versus adult (Haemonetics, ATSBC1ESE) PC storage containers. The influence of agitation on neonatal PC storage was evaluated. RESULTS:Results indicate neonatal PCs experience significantly higher O2 availability compared to adult PCs and can withstand greater insult to their ambient O2 concentration. Adjusting the agitation frequency of neonatal PCs stored from 20 to 400 rpm had no detrimental effect on O2 availability, compared to storage at 60 rpm. CONCLUSION:Neonatal PCs can maintain higher O2 availability and tolerate reduced agitation without compromising oxygenation; therefore, reduced agitation strategies may be a feasible option to minimize shear during storage without compromising O2 availability.

OBJECTIVE:Despite an increased cerebrovascular disease risk, the impact of Polycystic Ovary Syndrome (PCOS) on cerebrovascular haemodynamics and function is unknown. This study characterised cerebrovascular haemodynamics and function in women with PCOS versus healthy controls. DESIGN:Case-control study. PATIENTS:Fifteen women with PCOS (age: 31 ± 6 years; body mass index (BMI): 31.8 ± 5.7 kg/m2) and 16 controls (age: 30 ± 7 years; BMI: 29.9 ± 5.5 kg/m2). MEASUREMENTS:Resting global cerebral blood (CBF) was assessed by 3T MRI. Middle- and posterior cerebral artery blood velocities (MCAv, PCAv) were measured by Doppler ultrasound and pulsatility index (MCAPI, PCAPI) calculated. Neurovascular coupling (NVC), internal carotid artery cerebrovascular reactivity (CVRCO2) and dynamic cerebral autoregulation (dCA) directional sensitivity were assessed using a visual stimulus, 6% fixed-inspired CO2 and repeated squat-stand manoeuvres, respectively. RESULTS:Resting CBF (PCOS: 57.2 ± 7.5 ml/100 g/min; controls: 61.6 ± 11.6 ml/100 g/min, p = 0.25) and MCAv, PCAv, MCAPI and PCAPI (all p > 0.05) were similar between groups. NVC (14 ± 4.9% vs. 13 ± 3.4%, p = 0.45), CVRCO2 (5.1 ± 1.9% vs. 6.5 ± 2.9%, p = 0.20) and dCA directional sensitivity were similar between groups. However, women with PCOS had elevated relative PCAPI during NVC (PCOS: 12.0 ± 5.6% vs. controls: 7.0 ± 3.8%, p = 0.04), and impaired vasodilation of the internal carotid artery during CVRCO2 (PCOS: -0.10 ± 0.22 mm vs. controls: 0.18 ± 0.24 mm, p < 0.01). CONCLUSIONS:Cerebrovascular function is largely preserved in women with PCOS, although elevated arterial pulsatility and impaired vasodilatory response to carbon dioxide may indicate early endothelial dysfunction in the cerebral vasculature. Larger studies are needed to confirm this in view of our limited study power.

BACKGROUND:Storage of platelets as platelet concentrates for transfusion is limited to 7 days in the United Kingdom due to deleterious effects on platelet quality and function that occur over time. Oxygen (O2) availability and sufficient gaseous exchange are known to be essential in maintaining the viability and function of platelets stored for transfusion. Despite this, there is a paucity of studies undertaking direct measures of O2 and optimization of conditions throughout storage. We address this and modulate the storage conditions to improve platelet quality and function. STUDY DESIGN AND METHODS:Electron paramagnetic resonance oximetry was implemented to directly measure the [O2] experienced by stored platelet concentrates and the O2 consumption rate under standard blood banking conditions. From these direct measures the mathematical modeling was then applied to predict the main parameters contributing to effective O2 distribution throughout the unit. RESULTS:This study demonstrates reducing the storage [O2] to reflect near physiological levels significantly alters O2 distribution within the unit and negatively impacts platelet functionality and quality, and therefore is not a viable storage option. DISCUSSION:We show the reduction of platelet concentration within a unit improves O2 availability and pH, promotes a more uniform distribution of O2 throughout prolonged storage, and maintains platelet agonist-induced aggregation comparable to 100% platelet concentration. This may be a viable option and could potentially lead to reduced donor demand.

The impact of the biophysical environment on the platelet storage lesion (PSL) has mainly focused on reduced temperature storage, overlooking the significance of storage-induced shear stress. Shear stress in platelet storage refers to the frictional force acting parallel to the bag surface and exists solely through the implementation of agitation. This study investigates whether minimizing exposure to agitation-induced shear stress can alleviate the unexplained loss of function in stored platelet concentrates for neonatal transfusion (neonatal PCs). Using particle tracking analysis, fluid motion was measured in neonatal and adult platelet storage bags under agitation frequencies ranging from 20–60 rpm. Platelets stored at 20-60 rpm agitation over 8 days were examined by biochemical analysis, aggregation, and expression of activation markers. Results indicate that neonatal PCs experience significantly higher storage-induced shear stress compared to adult doses, leading to reduced functionality and increased activation from day 2 of storage. Adjusting the neonatal PC agitation frequency to 20 rpm improved functionality in early storage, while 40 rpm maintains this improvement throughout storage with reduced activation, compared to 60 rpm storage. This study confirms that small volume PC storage for neonatal use contributes to the PSL through the induction of shear stress, suggesting further evaluation of the recommended agitation frequency for neonatal PCs or postponement of the production of neonatal PCs until requested for neonatal transfusion.

BACKGROUND:Platelet derived extracellular vesicles (EVs) display a pro-coagulant phenotype and are generated throughout platelet concentrate (PC) storage. Cold storage (CS) of PCs is thought to provide a superior haemostatic advantage over room temperature (RT) storage and could prolong the storage time. However, the effect of storage conditions on EV generation and PC function is unknown. We investigated EV production under CS and RT conditions and assessed whether these EVs exhibited a more pro-coagulant phenotype in model experiments.MATERIALS AND METHODS:Buffy-coat-derived PCs in a platelet additive solution (PAS) to plasma ratio of approximately 65:35 were stored at RT (22 ± 2°C) or CS (4 ± 2°C) for a prolonged storage duration of 20 days. Impedance aggregometry assessed platelet function. EVs were isolated throughout storage and quantified using nanoparticle tracking analysis. EVs were applied to a coagulation assay to assess the impact on fibrin clot formation and lysis.RESULTS:CS produced significantly larger EVs from day 4 onwards. EV concentration was significantly increased in CS compared to RT from day 15. EVs, regardless of storage, significantly reduced time to clot formation and maximum optical density measured compared to the no EV control. Clot formation was proportionate to the number of EV applied but was not statistically different across storage conditions when corrected for EV number.CONCLUSION:EVs in CS and RT units showed similar clot formation capacity. However, the higher number of larger EVs generated in CS compared to RT suggests PC units derived from CS conditions may overall exhibit a haemostatically superior capacity compared to RT storage.

Lipoprotein-apheresis (apheresis) removes LDL-cholesterol in patients with severe dyslipidemia. However, reduction is transient, indicating that the long-term cardio-vascular benefi ts of apheresis may not solely be due to LDL removal. Microparticles (MPs) are submicron vesicles released from the plasma membrane of cells. MPs, particularly platelet-derived MPs, are increasingly being linked to the pathogenesis of many diseases. We aimed to characterize the effect of apheresis on MP size, concentration, cellular origin

INTRODUCTION:Passive immunotherapy using polyclonal antibodies plays an important role in preventing and treating antigenic and pathogenic diseases. Polyclonal antibodies are used for therapeutic, diagnostic and investigational purposes, with adjuvants employed to enhance the immune response against proteins that are poorly antigenic or self-antigens. This study aimed to optimize current immunization methods by evaluating the novel adjuvant CoVaccine HT™ against the established Freund's at producing ovine polyclonal antibodies against pro-inflammatory cytokine human recombinant tumor necrosis factor alpha (TNF-α). METHODS:Castrated male Aberfield cross sheep were immunized with TNF-α in CoVaccine HT™ or Freund's adjuvant. The binding titer of antibodies for TNF-α and neutralization titer were determined in vitro, as well as the strength of antibody binding by a simple small scale affinity chromatography elution experiment. Animal welfare was monitored through inspection of immunization site reactions at regular time points and graded according to reaction size. The second part of the study looked at re-immunization using Freund's adjuvant alone every 4- or 8-weeks. RESULTS:Freund's generated significantly higher antibody binding titers than CoVaccine HT™ but were less effective at neutralizing TNF-alpha which is a better indicator of functional potency. CoVaccine HT™ also caused fewer immunization site reactions, while no statistical difference was observed in the binding strength of antibodies. Re-immunization every 4- and 8-weeks showed no statistical difference. CONCLUSION:This study provides evidence that CoVaccine HT™ is superior to Freund's adjuvant for the production of antibodies to TNF-α, and supports the use of this alternative adjuvant for clinical and experimental use. The outcomes gained through this study are applicable to passive and active immunotherapy for the generation of polyclonal antibodies in human and veterinary medicine.

The coronavirus, COVID-19 pandemic spread across the globe in 2020, with an initial high case mortality in those requiring intensive care treatment due to serious complication. A vaccine programme was quickly developed and currently the UK is one of highest double vaccinated and boosted countries in the world. Despite tremendous efforts by the UK, new cases of COVID-19 are still occurring, due to viral mutation. A major problem associated with COVID-19 is the large a-symptomatic spread within the population. Little investigation into the a-symptomatic population has been carried out and therefore we pose that the residual effects of a-symptomatic infection is still largely unknown. Prior to mass vaccination, a multi-phased single cohort study of IgM and IgG COVID-19 antibody prevalence and the associated haemostatic changes were assessed in a Welsh cohort of 739 participants, at three time points. Positive antibody participants with age and gender matched negative antibody controls were assessed at 0, 3 and 6 months. Antibody positive females appeared to have lower antibody responses in comparison to their a-symptomatic male counterparts. Despite this initial testing showed a unique significant increase in TRAP-6-induced platelet aggregation, prothrombin time (PT) and clot initiation time. Despite coagulation parameters beginning to return to normal at 3 months, significant decreases are observed in both haemoglobin and haematocrit levels. The production of extracellular vesicles (EV) was also determined in this study. Although the overall number of EV does not change throughout the study, at the initial 0 months' time point a significant increase in the percentage of circulating pro-coagulant platelet derived EV is seen, which does not appear to be related to the extent of platelet activation in the subject. We conclude that early, but reversible changes in haemostatic pathways within the a-symptomatic, female, antibody positive COVID-19 individuals are present. These changes may be key in identifying a period of procoagulative risk for a-symptomatic female patients.

The methodologies described in this chapter inform on how to incorporate extracellular vesicles (EV) in model systems to investigate their role in the initiation and progression of the atherosclerotic plaque. The section will cover application of EV in coagulation and thrombus formation, monocytic migration, and adhesion to endothelial monolayers. These methodologies can be used with EV isolated from any cell type and under any conditions.

Background: Extracellular vesicles (EVs) derived from endothelial cells are elevated in cardiovascular disease and promote inflammation and coagulation. Hypoxia is often a key feature and is itself a potent stimulator of increased EV production. Inorganic nitrite (NO2-) has beneficial and protective effects that are enhanced in hypoxia. Objectives: Investigate the impact of hypoxia on the functional capacity of EV derived from endothelial cells under hypoxia, and assess whether pre-treatment of endothelial cells with NO2- can alter EV function. Methods: Differential ultracentrifugation was used to isolate EV from the cultured endothelial cell line HECV (CEV), and from primary human umbilical cord derived endothelial cells (PEV), with time-resolved fluorescence used to assess EV protein composition. Clot formation was induced by thrombin and calcium in two assays; using an Alexa Fluor 594 human fibrinogen conjugate assay and standard turbidometry. Platelet aggregation was determined using multiple electrode aggregometry. Scanning electron microscopy was used to visualise fibrin clots. Results: Hypoxia exposure (1% O-2) significantly increased CEV production in comparison to normoxia (21% O-2) (1825 +/- 72 EVs/cell vs 117 +/- 9 EVs/cell, p < 0.001, respectively) but had no effect on CEV mean size (221 +/- 6 nm vs 203 +/- 4 nm, p > 0.05). Hypoxia-derived PEVs contained significantly more tissue factor than normoxiaderived EVs (Relative Fluorescence Units (RFU) = 7666 +/- 1698 vs 5958 +/- 1644, p < 0.001, respectively) and less tissue factor pathway inhibitor (RFU = 9799 +/- 2353 vs 19723 +/- 2698, p < 0.05). Hypoxia significantly increased CEV induced fibrin polymer formation compared to normoxia (% area = 46.98 +/- 0.97 vs 36.36 +/- 0.72, p < 0.05). Pre-treatment of endothelial cells with NO2- in hypoxia abrogated this effect (% area = 15.70 +/- 1.99, p < 0.001). Hypoxia derived CEV non-significantly increased the maximum clot formed, shortened time to max clot, and increased time to clot lysis by turbidometry. ADP-mediated platelet aggregation was significantly elevated with PEV derived from hypoxia compared to normoxia (888.0 +/- 32.2 AU*min vs 671.5.2 +/- 28.3 AU*min, p < 0.01). This was abrogated by pre-treatment of hypoxic endothelial cells with NO2- (716.5 +/- 744.3 AU*min, p < 0.001). Conclusions: Hypoxia-derived PEVs and CEVs exhibit increased procoagulant activity compared to normoxiaderived EVs, which we confirm to be mediated by an imbalance of TF/TFPI. Pre-treatment of endothelial cells with NO2- reduces the pro-coagulant activity of EVs via a mechanism that is Hypoxia-inducible factor 1 (HIF-1) dependent, but independent of TF/TFPI.

Extracellular vesicles (EVs) are nanometre-sized vesicles released from most cells, including adipocytes. Relatively little is known about adipocyte-derived EVs (ADEVs) in comparison to other EV subtypes, though interest in ADEVs as potential paracrine and endocrine communicators of adipose tissue in obesity is building. Current evidence indicates that ADEVs contribute to the development of adipose tissue dysfunction; a key feature of obese adipose tissue that it is associated with obesity-related comorbidities including cardiovascular disease (CVD). This review summarises our current knowledge of ADEVs in the development of adipose tissue dysfunction and the potential of ADEVs to disrupt redox signalling and exert vascular effects that may exacerbate CVD in obesity.

Introduction: Polycystic Ovary syndrome (PCOS) is a metabolic disorder associated with increased cardiovascular disease risk. Exercise is an effective treatment strategy to manage symptoms and reduce long-term health risk. High-intensity interval training (HIIT) has been suggested as a more efficient exercise mode in PCOS; however, it is not clear whether HIIT is superior to moderate intensity steady state exercise (MISS). Methods: We synthesized available data through a systematic review and meta-analysis to compare the effectiveness of isolated HIIT and MISS exercise interventions. Our primary outcome measures were cardiorespiratory fitness and insulin resistance, measured using V˙ O 2max and HOMA-IR respectively. Results: A total of 16 studies were included. Moderate-quality evidence from 16 studies identified significant improvements in V˙ O 2max following MISS (Δ = 1.081 ml/kg/min, p < 0.001, n = 194), but not HIIT (Δ = 0.641 ml/kg/min, p = 0.128, n = 28). Neither HIIT nor MISS improved HOMA-IR [(Δ = −0.257, p = 0.374, n = 60) and (Δ = −0.341, p = 0.078, n = 159), respectively]. Discussion: A significant improvement in V˙ O 2max was evident following MISS, but not HIIT exercise in women with PCOS. This contrasts with previous literature in healthy and clinical cohorts that report superior benefits of HIIT. Therefore, based on available moderate-quality evidence, HIIT exercise does not provide superior outcomes in V˙ O 2max compared with MISS, although larger high-quality interventions are needed to fully address this. Additional dietary/pharmacological interventions may be required in conjunction with exercise to improve insulin sensitivity.

Background and aims: Obesity is associated with an increased risk of cardiovascular disease, but the mechanisms involved are not completely understood. In obesity, the adipocyte microenvironment is characterised by both hypoxia and inflammation. Therefore, we sought to determine whether extracellular vesicles (EVs) derived from adipocytes in this setting might be involved in mediating cardiovascular disease, specifically by promoting leukocyte attachment to vascular endothelial cells. Methods: Mature 3T3-L1 adipocytes were incubated for 24 h under control, TNF-alpha (30 ng/mL), hypoxia (1% O-2), or TNF-alpha+ hypoxia (30 ng/mL, 1% O-2) conditions. EVs were isolated by differential ultracentrifugation and analysed by nanoparticle tracking analysis. Primary human umbilical vein endothelial cells (HUVECs) were treated with EVs for 6 h before being lysed for Western blotting to investigate changes in adhesion molecule production, or for use in leukocyte attachment assays. Results: EVs from adipocytes treated with TNF-alpha and TNF-alpha+ hypoxia increased vascular cell adhesion molecule (VCAM-1) production in HUVECs compared to basal level (4.2 +/- 0.6 and 3.8 +/- 0.3-fold increase, respectively (p < 0.05)), an effect that was inhibited by an anti-TNF-alpha neutralising antibody. Production of other adhesion molecules (E-selectin, P-selectin, platelet endothelial cell adhesion molecule and VE-Cadherin) was unchanged. Pre-incubating HUVECs with TNF-alpha+ hypoxia EVs significantly increased leukocyte attachment compared to basal level (3.0 +/- 0.4-fold increase (p < 0.05)). Conclusions: Inflammatory adipocyte EVs induce VCAM-1 production in vascular endothelial cells, accompanied by enhanced leukocyte attachment. Preventing adipocyte derived EV-induced VCAM-1 upregulation may offer a novel therapeutic target in the prevention of obesity-driven cardiovascular disease.

Adipocyte-derived extracellular vesicles (EVs) may serve as novel endocrine mediators of adipose tissue and impact upon vascular health. However, it is unclear whether adipocyte-derived EVs are present in the human circulation. Therefore, the purpose of this study was to seek evidence for the presence of adipocyte-derived EVs in circulating plasma. Size-exclusion chromatography of platelet-free plasma identified fractions 5 to 10 as containing EVs by a peak in particle concentration, which corresponded with the presence of EV and adipocyte proteins. Pooling fractions 5 to 10 and subjecting to ultracentrifugation yielded a plasma EV sample, as verified by transmission electron microscopy (TEM) showing EV structures and Western blotting for EV (e.g., CD9 and Alix) and adipocyte markers. Magnetic beads and a solid-phase assay were used to deplete the EV sample of the four major families of circulating EVs: platelet-derived, leukocyte-derived, endothelial-derived, and erythrocyte-derived EVs. Postdepletion samples from both techniques contained EV structures as visualized by TEM, as well as CD9, Alix, and classic adipocyte proteins. Postdepletion samples also contained a range of other adipocyte proteins from an adipokine array. Adipocyte proteins and adipokines are expressed in optimally processed plasma EV samples, suggesting that adipocyte-derived EVs are secreted into the human circulation.

Abstract Extracellular vesicles (EVs) are implicated in the pathogenesis of cardiovascular disease (CVD). Specifically, platelet-derived EVs are highly pro-coagulant, promoting thrombin generation and fibrin clot formation. Nitrate supplementation exerts beneficial effects in CVD, via an increase in nitric oxide (NO) bioavailability. Clopidogrel is capable of producing NO-donating compounds, such as S-nitrosothiols (RSNO) in the presence of nitrite and low pH. The aim of this study was to assess the effect of nitrate supplementation with versus without clopidogrel therapy on circulating EVs in coronary artery disease (CAD) patients. In this randomized, double-blind, placebo-controlled study, CAD patients with (n = 10) or without (n = 10) clopidogrel therapy received a dietary nitrate supplement (SiS nitrate gel) or identical placebo. NO metabolites and platelet activation were measured using ozone-based chemiluminescence and multiple electrode aggregometry. EV concentration and origin were determined using nanoparticle tracking analysis and time-resolved fluorescence. Following nitrate supplementation, plasma RSNO was elevated (4.7 ± 0.8 vs 0.2 ± 0.5 nM) and thrombin-receptor mediated platelet aggregation was reduced (−19.9 ± 6.0 vs 4.0 ± 6.4 U) only in the clopidogrel group compared with placebo. Circulating EVs were significantly reduced in this group (−1.183e11 ± 3.15e10 vs −9.93e9 ± 1.84e10 EVs/mL), specifically the proportion of CD41+ EVs (−2,120 ± 728 vs 235 ± 436 RFU [relative fluorescence unit]) compared with placebo. In vitro experiments demonstrated clopidogrel–SNO can reduce platelet-EV directly (6.209e10 ± 4.074e9 vs 3.94e11 ±  1.91e10 EVs/mL). In conclusion, nitrate supplementation reduces platelet-derived EVs in CAD patients on clopidogrel therapy, increasing patient responsiveness to clopidogrel. Nitrate supplementation may represent a novel approach to moderating the risk of thrombus formation in CAD patients.

Dietary nitrate supplementation has been shown to increase nitric oxide (NO) metabolites, reduce blood pressure (BP) and enhance exercise performance. Acute exposure to ultraviolet (UV)-A light also increases NO bioavailability and reduces BP. We conducted a randomized, counterbalanced placebo-controlled trial to determine the effects of UV-A light alone and in combination with nitrate on the responses to sub-maximal steady-state exercise and time trial (TT) performance. Nine cyclists (VO2max 53.1 ± 4.4 ml/kg/min) completed five performance trials comprising 10 min submaximal steady-state cycling followed by a 16.1 km TT. Following a familiarization the final four trials were preceded, in random order, by either 1) Nitrate gels (NIT) + UV-A, 2) Placebo (PLA) + UV-A, 3) NIT + Sham light (SHAM) and 4) PLA + SHAM (control). The NIT gels (2 x 60 ml gels, ~500 mg nitrate) or a nitrate-depleted PLA were ingested 2.5 h prior to the trial. The light exposure consisted of 20J/cm whole body irradiation with either UV-A or SHAM light. Plasma nitrite was measured preand postirradiation and VO2 was measured continuously during steady-state exercise. Plasma nitrite increased following NIT + SHAM (332 (292 – 377) nM; P=0.029) and NIT + UV-A (456 (312 – 666) nM; P=0.014) compared to PLA + SHAM (215 (167 – 277) nM). Differences between PLA + SHAM and PLA + UV-A (282 (248 – 356) nM) were small and nonsignificant. During steady state exercise VO2 was reduced following NIT + UVA (P=0.034) and tended to be lower in NIT + SHAM (P=0.086) but not PLA + UV-A (P=0.381) compared to PLA + SHAM.TT performance was significantly faster following NIT + UV-A, (1447 ± 41 s; P=0.005; d=0.47) but not PLA + UV-A (1450 ± 40 s; d=0.41) or NIT + SHAM (1455 ± 47 s; d=0.28) compared to PLA + SHAM. These findings demonstrate that exposure to UV-A light alone does not alter the physiological responses to exercise or improve performance a laboratory setting. A combination of UV-A and NIT, however, does improve cycling TT performance in this environment which may be due to a larger increase in NO availability.