DiMeN Doctoral Training Partnership: A Role for Neutrophils in Blood Clot Structure and Function: Thrombosis and Neutrophil Extracellular Traps (NETs)

"Blood clots that cause heart attacks and stroke are largely composed of fibrin, platelets and red blood cells. However, white blood cells are also incorporated into the clot, and their role in clot architecture and stability is poorly understood. Recent studies have shown neutrophils are capable of extruding their DNA and histones, producing an extracellular fibrous network that traps bacteria and pathogens (Neutrophil Extracellular Traps). Platelets have been implicated in NETs formation and NETs have been found in arterial and venous thrombi. Currently it is not fully known how these NETs interact with the fibrin network and how NETs influence formation of the clot, its function and its stability. This project will focus on the 1) Interaction of neutrophils with fibrin and the consequences of this interaction on clot formation and stability, 2) Role of NETs in regulating fibrin elastic properties and resistance to fibrinolysis, and 3) Investigation of how NETs incorporate into the fibrin network.

Neutrophil interactions with fibrin(ogen) will be investigated at a single molecule interaction level by AFM force spectroscopy. Fibrinogen will be coupled to AFM tips and used to probe neutrophils. We will also probe neutrophil fibrinogen interactions with optical tweezers set up in our laboratory. Neutrophils will be activated with C5a, LPS or PMA prior to probing of fibrin(ogen) binding. Binding kinetics will be investigated using plate binding assays. The role of NETs in regulating clot elastic properties (Young’s modulus, G’, and loss tangent, G’’) will be investigated using magnetic tweezers. Fibrin clots will be made from fibrinogen and plasma in the presence of PMA stimulated neutrophils to induce NETs formation. Fibrinolysis rates will be analysed in the presence of tPA using laser scanning confocal microscopy and turbidity measurements. The effect of NETs on fibrin intrafibrillar structure will be investigated using turbidimetric analysis of protofibril packing and high resolution cold-field scanning electron microscopy. Fibrin network structure will be investigated by confocal microscopy and permeation analysis. Finally, thrombi will be obtained from a murine vena cava thrombosis model and analysed for fibrin and NETs using immunohistochemical analysis and confocal microscopy.

The primary supervisor is based in the School of Medicine in Leeds and is an expert in vascular biology with particular focus on clot structure and function. The secondary supervisor is based in the School of Medicine in Sheffield and is a leukocyte biologist. The third supervisor is based in the School of Physics in Leeds and is an expert in biophysical analysis of fibrin. The fourth supervisor is based in Leeds and is a translational medicine expert.

The role of neutrophils and NETs in thrombosis provides an important link between inflammation and blood coagulation but is hitherto poorly understood. These PhD studies will elucidate the role of neutrophils and NETs in clot formation and may lead to the identification of novel therapeutic targets for the treatment of thrombosis."