Re: Modification and validation of a human endothelialised in-vitro thrombosis model to promote end-user uptake

Sarah’s award will enable the transfer two in vitro endothelialised thrombosis models, originally developed with an NC3Rs-BHF studentships award, to two labs where they will be optimised and tested for their fit-for-purpose in platelet and stroke research. Mouse models of thrombosis are widely used but lack standardisation, have limited human relevance and often use large numbers of animals to detect biologically relevant effects. Sarah will partner with researchers at the University of Reading to transfer her in vitro endothelialised model, designed to measure endothelial contributions to thrombus formation and antithrombotic efficacy, so that it can be adapted to incorporate a different fluidic system commonly used in the platelet research field. She will also collaborate with researchers at the University of Manchester to transfer her complex in vitro model of ferric chloride thrombosis to replace some mouse studies of ferric chloride-induced middle cerebral artery occlusion in the testing of thrombolytics.

Arterial thrombosis leading to myocardial infarction and strokes is a leading cause of death globally. Thrombi (blood clots) develop in arteries when the endothelial lining is disrupted, exposing extracellular matrix proteins, and releasing tissue factor. This leads to blood platelets adhering to the site of damage, becoming activated and recruiting more platelets. In parallel, the coagulation cascade is activated, leading to stabilisation of the platelet rich clot through the production of fibrin. The main therapeutic approach in the prevention of myocardial infarction and strokes, is antiplatelet therapy, which reduces the risk by ~25%. Current treatments however are limited by high patient variability and significant risk of bleeding. When patients are admitted to hospital with an occlusive thrombus, the only pharmacological approach to remove clots are thrombolytics (clot busting drugs) such as Alteplase, however these drugs are only useful within the first few hours of onset, and they pose a significant bleeding risk. There is therefore a significant drive for safer and more efficacious drugs for both the prevention and treatment of myocardial infarction and stroke.

To develop and test new antiplatelet and thrombolytic drugs, animal models of thrombosis are used extensively. These models are nonrecovery and involve damage to the blood vessel wall of an anaesthetised mouse using ferric chloride (FeCl3) or laser injury, and measurement of thrombus formation using Doppler or intravital microscopy. The models are limited by lack of standardisation and reproducibility, with large numbers of animals required to provide statistical power. Translation to humans is also limited due to significant species differences in platelet receptor expression, vessel phenotype and haemodynamic profiles.

We have previously developed replacements for in-vivo thrombosis models using endothelialised flow chambers as part of a BHF/NC3Rs studentship. We developed and characterised a basic model (prototype 1) to investigate endothelial contributions to thrombosis and antithrombotic efficacy, and a more sophisticated model (prototype 2), designed as a human in-vitro model of coronary artery thrombosis to replicate the mouse FeCl3 thrombosis model. We have identified two laboratories, keen to adopt the models, however, to meet the end user’s needs, the existing prototypes require modifications and
subsequent validation. The aims of the current proposal are to 1) Adapt and characterise prototype 1 using the Cellix fluidic system (used widely in the platelet field), to facilitate uptake at The University of Reading. This will replace animal experiments to investigate antithrombotic drugs which target the endothelium. 2) Adapt prototype 2 to mimic middle cerebral artery (MCA) thrombosis, using brain endothelial cells and haemodynamic conditions representative of human middle cerebral arteries. This will facilitate adoption by the University of Manchester to test novel hypothesise and thrombolytics
to replace the mouse FeCl3 stroke model, where appropriate.

Adaptation and validation of our endothelialised thrombosis prototypes, will enable adoption of the models by two leading institutions, which currently use in-vivo thrombosis models, delivering immediate impact on animal usage. The adaptations will also promote wider 3Rs impact, by providing an endothelialised model validated for use with a fluidic system common to most platelet groups; and by extending the application of the model to also
investigate stroke and test novel thrombolytics.