Characterisation, development and application of a clinical model of thrombosis and fibrinolysis

The demonstration of antithrombotic efficacy in man is challenging. Most techniques evaluate specific plasma or cellular components under static conditions in vitro. In contrast, in vivo thrombus initiation and growth occur in whole blood, under conditions of continuous flow and in the presence of vascular injury. An in vivo model for use in clinical studies presents significant safety issues and does not currently exist. The Badimon chamber is an ex vivo model of thrombosis that is suitable for use in clinical studies and has previously been used to assess novel antithrombotic regimens. Although well-established, previous characterisation studies were performed in a porcine system and using methodology that has since been superceded. In addition, it has a number of disadvantages that limit its broader applicability and has not previously been used to assess fibrinolysis. Having established The Badimon Chamber within my own institution, I developed the methodology and performed careful validation and characterisation studies with a particular emphasis on reproducibility. These developments allowed more efficient data analysis and the accurate addition of compounds to the extracorporeal circuit, both of which broaden the applicability of the technique. In subsequent studies, using a series of double-blind randomised controlled crossover studies in healthy volunteer cohorts, I utilised the updated methodology to address questions in separate but overlapping areas of cardiovascular medicine. The dynamic regulation of intravascular thrombus formation by the endogenous fibrinolytic system is central to the pathogenesis of acute atherosclerotic events, particularly within the coronary circulation. Previous work within our institution has provided novel insights into the role of endogenous fibrinolysis. Despite a growing body of evidence, a key limitation of studies to date is that the effects of acute endogenous t-PA release on in situ thrombus formation have not been demonstrated. Is endogenous endothelial t-PA released under agonist stimulation functionally active and able to enhance fibrinolysis of in situ thrombus? Firstly, I demonstrated that the addition of exogenous t-PA into the extracorporeal circuit of The Badimon Chamber results in a dose dependent increase in plasma D-dimer associated with a dose dependent reduction in thrombus formation, consistent with enhanced fibrinolysis. Having validated the model, I proceeded to investigate whether freshly released endogenous t-PA would have similar effects to exogenous t-PA. By combining intraarterial infusion of bradykinin into the human forearm in order to stimulate acute release of endogenous t-PA with an assessment of thrombus formation in the Badimon Chamber, I demonstrated that endogenous t-PA released acutely from the human vascular endothelium enhances fibrinolysis and limits in situ thrombus formation. These data validate the forearm model as a relevant model with which to assess acute fibrinolytic capacity, confirm the functional significance of t-PA released during agonist stimulation and suggest that further studies to explore its therapeutic manipulation are warranted. I went on to evaluate a promising small molecule PAI-1 inhibitor, PAI-749, using assessments of ex vivo thrombosis complimented by extensive in vitro studies. Interestingly, in contrast to the promising results seen with this compound in preclinical models, we were unable to demonstrate efficacy in any of the clinical models used, highlighting the potential pitfalls of relying solely on in vitro and preclinical models during early compound development. In the final phase of this work, I used the chamber to explore the prothrombotic effects of exposure to air pollution. A plethora of observational data exist to suggest that acute exposure to particulate air pollution can trigger vascular events including myocardial infarction although the underlying mechanisms are only partly understood. Using a unique human exposure facility, we demonstrated that inhalation of diesel exhaust causes platelet activation and enhances thrombus formation. These data provide a plausible mechanism linking exposure to particulate air pollution with acute cardiovascular events including myocardial infarction. Furthermore, in a separate study we were able to demonstrate that reducing the particulate component of the exposure using a commercially available particle trap prevents the detrimental effects on ex vivo thrombosis and endothelial function. These data support calls for the application of particle traps to diesel-powered vehicles in order to limit a range of adverse cardiovascular effects that result from exposure to traffic-derived air pollution

Role of shear stress and tPA concentration in the fibrinolytic potential of thrombi

Funding: This research was funded by British Heart Foundation PG/08/127/26517 awarded to N.A.B. and D.E.N. C.S.W. and N.J.M. were supported by the British Heart Foundation project grants (PG/15/82/31721 and PG/20/17/35050). D.E.N. and A.J.L. were supported by the British Heart Foundation project grant PG/04/131/18118. D.E.N. is supported by the British Heart Foundation (CH/09/002, RE/18/5/34216, RG/16/10/32375) and is the recipient of a Wellcome Trust Senior Investigator Award (WT103782AIA).Peer reviewedPublisher PD

Fibrin clot structure remains unaffected in young, healthy individuals after transient exposure to diesel exhaust

Exposure to urban particulate matter has been associated with an increased risk of cardiovascular disease and thrombosis. We studied the effects of transient exposure to diesel particles on fibrin clot structure of 16 healthy individuals (age 21- 44). The subjects were randomly exposed to diesel exhaust and filtered air on two separate occasions. Blood samples were collected before exposure, and 2 and 6 hours after exposure. There were no significant changes on clot permeability, maximum turbidity, lag time, fibre diameter, fibre density and fibrinogen level between samples taken after diesel exhaust exposure and samples taken after filtered air exposure. These data show that there are no prothrombotic changes in fibrin clot structure in young, healthy individuals exposed to diesel exhaust

Analysis of Nigerians with Apparently Sporadic Parkinson Disease for Mutations in LRRK2, PRKN and ATXN3

Several genetic variations have been associated with Parkinson disease in different populations over the past few years. Although a considerable number of worldwide populations have been screened for these variants, results from Sub-Saharan populations are very scarce in the literature. In the present report we have screened a cohort of Parkinson disease patients (n = 57) and healthy controls (n = 51) from Nigeria for mutations in the genes PRKN, LRRK2 and ATXN3. No pathogenic mutations were found in any of the genes. Hence, common pathogenic mutations in these genes, observed in several different populations, are not a frequent cause of Parkinson disease in Nigeria

Fire Simulation and Cardiovascular Health in Firefighters

BACKGROUND: Rates of myocardial infarction in firefighters are increased during fire suppression duties, and are likely to reflect a combination of factors including extreme physical exertion and heat exposure. We assessed the effects of simulated fire suppression on measures of cardiovascular health in healthy firefighters. METHODS: In an open-label randomized crossover study, 19 healthy firefighters (age, 41±7 years; 16 males) performed a standardized training exercise in a fire simulation facility or light duties for 20 minutes. After each exposure, ex vivo thrombus formation, fibrinolysis, platelet activation, and forearm blood flow in response to intra-arterial infusions of endothelial-dependent and -independent vasodilators were measured. RESULTS: After fire simulation training, core temperature increased (1.0±0.1°C) and weight reduced (0.46±0.14 kg, P<0.001 for both). In comparison with control, exposure to fire simulation increased thrombus formation under low-shear (73±14%) and high-shear (66±14%) conditions (P<0.001 for both) and increased platelet-monocyte binding (7±10%, P=0.03). There was a dose-dependent increase in forearm blood flow with all vasodilators (P<0.001), which was attenuated by fire simulation in response to acetylcholine (P=0.01) and sodium nitroprusside (P=0.004). This was associated with a rise in fibrinolytic capacity, asymptomatic myocardial ischemia, and an increase in plasma cardiac troponin I concentrations (1.4 [0.8–2.5] versus 3.0 [1.7–6.4] ng/L, P=0.010). CONCLUSIONS: Exposure to extreme heat and physical exertion during fire suppression activates platelets, increases thrombus formation, impairs vascular function, and promotes myocardial ischemia and injury in healthy firefighters. Our findings provide pathogenic mechanisms to explain the association between fire suppression activity and acute myocardial infarction in firefighters. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01812317

Lipopolysaccharide and Tumor Necrosis Factor Regulate Parkin Expression via Nuclear Factor-Kappa B

Inflammation and oxidative stress have been implicated in the pathophysiology of Parkinson's disease (PD) and inhibition of microglial activation attenuates degeneration of dopaminergic (DA) neurons in animal models of PD. Loss-of-function mutations in the parkin gene, which encodes an E3 ubiquitin ligase, cause autosomal recessive parkinsonism. While most studies on Parkin have focused on its function in neurons, here we demonstrate that Parkin mRNA and protein is detectable in brain-resident microglia and peripheral macrophages. Using pharmacologic and genetic approaches, we found that Parkin levels are regulated by inflammatory signaling. Specifically, exposure to LPS or Tumor Necrosis Factor (TNF) induced a transient and dose-dependent decrease in Parkin mRNA and protein in microglia, macrophages and neuronal cells blockable by inhibitors of Nuclear Factor-Kappa B (NF-κB) signaling and not observed in MyD88-null cells. Moreover, using luciferase reporter assays, we identified an NF-κB response element in the mouse parkin promoter responsible for mediating the transcriptional repression, which was abrogated when the consensus sequence was mutated. Functionally, activated macrophages from Parkin-null mice displayed increased levels of TNF, IL-1β, and iNOS mRNA compared to wild type macrophages but no difference in levels of Nrf2, HO-1, or NQO1. One implication of our findings is that chronic inflammatory conditions may reduce Parkin levels and phenocopy parkin loss-of-function mutations, thereby increasing the vulnerability for degeneration of the nigrostriatal pathway and development of PD

Effect of wood smoke exposure on vascular function and thrombus formation in healthy fire fighters

Background: Myocardial infarction is the leading cause of death in fire fighters and has been linked with exposure to air pollution and fire suppression duties. We therefore investigated the effects of wood smoke exposure on vascular vasomotor and fibrinolytic function, and thrombus formation in healthy fire fighters. Methods: In a double-blind randomized cross-over study, 16 healthy male fire fighters were exposed to wood smoke (~1 mg/m3 particulate matter concentration) or filtered air for one hour during intermittent exercise. Arterial pressure and stiffness were measured before and immediately after exposure, and forearm blood flow was measured during intra-brachial infusion of endothelium-dependent and -independent vasodilators 4–6 hours after exposure. Thrombus formation was assessed using the ex vivo Badimon chamber at 2 hours, and platelet activation was measured using flow cytometry for up to 24 hours after the exposure. Results: Compared to filtered air, exposure to wood smoke increased blood carboxyhaemoglobin concentrations (1.3% versus 0.8%; P &lt; 0.001), but had no effect on arterial pressure, augmentation index or pulse wave velocity (P &gt; 0.05 for all). Whilst there was a dose-dependent increase in forearm blood flow with each vasodilator (P &lt; 0.01 for all), there were no differences in blood flow responses to acetylcholine, sodium nitroprusside or verapamil between exposures (P &gt; 0.05 for all). Following exposure to wood smoke, vasodilatation to bradykinin increased (P = 0.003), but there was no effect on bradykinin-induced tissue-plasminogen activator release, thrombus area or markers of platelet activation (P &gt; 0.05 for all). Conclusions: Wood smoke exposure does not impair vascular vasomotor or fibrinolytic function, or increase thrombus formation in fire fighters. Acute cardiovascular events following fire suppression may be precipitated by exposure to other air pollutants or through other mechanisms, such as strenuous physical exertion and dehydration.Originally included in thesis in manuscript form.</p

Effect of remote ischaemic conditioning on clinical outcomes in patients with acute myocardial infarction (CONDI-2/ERIC-PPCI): a single-blind randomised controlled trial.

BACKGROUND: Remote ischaemic conditioning with transient ischaemia and reperfusion applied to the arm has been shown to reduce myocardial infarct size in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI). We investigated whether remote ischaemic conditioning could reduce the incidence of cardiac death and hospitalisation for heart failure at 12 months. METHODS: We did an international investigator-initiated, prospective, single-blind, randomised controlled trial (CONDI-2/ERIC-PPCI) at 33 centres across the UK, Denmark, Spain, and Serbia. Patients (age >18 years) with suspected STEMI and who were eligible for PPCI were randomly allocated (1:1, stratified by centre with a permuted block method) to receive standard treatment (including a sham simulated remote ischaemic conditioning intervention at UK sites only) or remote ischaemic conditioning treatment (intermittent ischaemia and reperfusion applied to the arm through four cycles of 5-min inflation and 5-min deflation of an automated cuff device) before PPCI. Investigators responsible for data collection and outcome assessment were masked to treatment allocation. The primary combined endpoint was cardiac death or hospitalisation for heart failure at 12 months in the intention-to-treat population. This trial is registered with ClinicalTrials.gov (NCT02342522) and is completed. FINDINGS: Between Nov 6, 2013, and March 31, 2018, 5401 patients were randomly allocated to either the control group (n=2701) or the remote ischaemic conditioning group (n=2700). After exclusion of patients upon hospital arrival or loss to follow-up, 2569 patients in the control group and 2546 in the intervention group were included in the intention-to-treat analysis. At 12 months post-PPCI, the Kaplan-Meier-estimated frequencies of cardiac death or hospitalisation for heart failure (the primary endpoint) were 220 (8·6%) patients in the control group and 239 (9·4%) in the remote ischaemic conditioning group (hazard ratio 1·10 [95% CI 0·91-1·32], p=0·32 for intervention versus control). No important unexpected adverse events or side effects of remote ischaemic conditioning were observed. INTERPRETATION: Remote ischaemic conditioning does not improve clinical outcomes (cardiac death or hospitalisation for heart failure) at 12 months in patients with STEMI undergoing PPCI. FUNDING: British Heart Foundation, University College London Hospitals/University College London Biomedical Research Centre, Danish Innovation Foundation, Novo Nordisk Foundation, TrygFonden

Have I Lost My Goggles in the Optical Coherence Tomography Pullback!?: A Very Unusual Finding in Coronary Optical Coherence&nbsp;Tomography

A case report of a very unusual finding in coronary optical coherence tomograph