Replacement of in vivo arterial thrombosis models using human placental arteries: The birth of a new approach

Platelets play a pivotal role in the initiation and development of arterial thrombosis, and as such antiplatelet treatments represent the cornerstone therapy for the prevention of myocardial infarction and stroke.  Current treatment regimens reduce the risk by ~25%, but patient variability is high, and bleeding remains a challenge. There is therefore a significant drive for safer and more efficacious antiplatelet drugs. To understand the basic biology of thrombosis and develop new antiplatelet drugs, animal models of thrombosis are extensively used globally. These models are non-recovery and involve damage to the blood vessel wall of an anaesthetised animal using ferric chloride (FeCl₃) or laser injury, and measurement of thrombus formation using 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. The aim of this project is to REPLACE in vivo thrombosis models with equivalent human ex vivo models. The models will comprise of human placental arteries, perfused with human blood at a physiological shear rate. Vascular damage will be initiated by FeCl₃ and laser injury, comparable to animal models and thrombus formation imaged using fluorescence microscopy. We have already successfully optimised methods for cold storage (21h post-delivery) and cryopreservation of placental vessels demonstrating maintained vessel viability and endothelial function, maximising accessibility. In this study, the models will be compared in fresh, cold stored (6h and 21h) and cryopreserved vessels. Results obtained using the human ex vivo model will be validated against equivalent published in-vivo studies testing standard antithrombotic drugs (aspirin, ticagrelor, apixaban) and the model will be established in two of the leading thrombosis groups in the UK (Prof Gibbins, University of Reading; Prof Naseem, University of Leeds) to test interlaboratory variability and further validation. The final part of the project will explore the potential of knocking down individual proteins separately in the artery, platelets and in both, using commercially available PROTACs . Development of human models with the capability to knockdown proteins will enable a larger proportion of in vivo experiments to be replaced yielding greater 3Rs impact. It would also facilitate investigations to elucidate platelet verses vessel contributions to thrombus formation, furthering mechanistic understanding. In summary, this project will deliver a more ethical and scientifically valid approach to investigate human arterial thrombosis, replacing current in vivo approaches.