Role of Neutrophils in Fibrin Structure and Function: How do Cells, Fibrin and Neutrophil Extracellular Traps (NETs) Integrate in Thrombi and Blood Cl

Blood clots that cause heart attacks and strokes are 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. Neutrophils are a type of white cell involved in innate immunity and are able to secrete a number of proteases such as elastases that are known to degrade fibrin. Furthermore, 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 or NETs). Platelets have been implicated in NETs formation and NETs have been found in arterial and venous thrombi.

<p>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. Moreover, recent studies have shown that fibrin interacts with red blood cells via a b3 integrin receptor, and with platelets via the a2bb3 and GPVI receptors. Fibrin further binds endothelial cells via integrin receptors. It is not known if fibrin also binds neutrophils, but fibrin is known to interact with NETs (Longstaff C, JBC 2013). 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 clot network in vivo.</p> <h3>Hypothesis:</h3> <p>The activation of neutrophils in the vicinity of fibrin formation leads to the incorporation of NETs into the fibrin network, which leads to a denser clot architecture, increases clot stiffness, reduces elastic properties of the clot, increases clot resistance to fibrinolysis and worsens thrombosis.</p> <h3>Aims:</h3> <p>The role of neutrophils and NETs in thrombosis provides an important link between inflammation and blood coagulation but is hitherto poorly understood. This PhD project 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.</p> <h3>Plan of Investigation:</h3> <p>The plan of investigation will focus on 3 main objectives: 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 clot network in vivo.</p> <p>Neutrophils will be obtained from blood by standard density gradient centrifugation methods. 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 using amine-coupling methods as previously described (Carvalho FA, ACS Nano 2010) and used to probe washed neutrophils. We will also probe neutrophil fibrinogen interactions with optical tweezers recently set up in our laboratory. In some of the experiments, fibrinogen will be converted to fibrin by bathing the tip in thrombin. In addition, 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.</p> <p>The role of NETs in regulating clot elastic properties (Young&rsquo;s modulus, G&rsquo;, and loss tangent, G&rsquo;&rsquo;) will be investigated using magnetic tweezers. Fibrin clots will be made from fibrinogen and from plasma in the presence of PMA stimulated neutrophils to induce NETs formation. Clot stiffness and viscous properties will be investigated using magnetic tweezers as previously described (Domingues M, Blood 2016; Allan P, JTH 2012). The candidate will also analyse the effects of NETs on fibrin intrafibrillar structure to investigate whether NETs influence protofibril packing of the fibres or whether NETs integrate with fibrin at the fibre level. Intrafibrillar structure will be investigated using turbidimetric analysis of protofibril packing and high-resolution cold-field scanning electron microscopy as previously described (Domingues M, Blood 2016). The effects on overall clot network structure will be investigated by confocal microscopy and permeation analysis. Finally, rates of fibrinolysis by tPA and plasminogen will be investigated using standard laser scanning confocal microscopy and turbidity measurements.</p> <p>The role of NETs interaction with fibrin during in vivo thrombosis will be investigated using murine models of thrombosis (Duval C, ATVB 2016). The candidate will develop a novel restricted blood flow thrombosis model based on ligation of the murine vena cava, which previously has been shown to induce thrombosis and fibrin deposition accompanied by inflammation and NETosis (Brill A, JTH 2012). The murine vena cava will be exposed through dissection under general anaesthesia. Ligatures will be placed around the vena cava, with a small-diameter hypodermic needle as spacer. The spacer will be removed and the ensuing thrombosis occurs over 3-5 days post-intervention. The animals will be sacrificed and the thrombi obtained for analysis of fibrin and NETs using immunohistochemical analysis. We will also explore the use of light sheet microscopy to image the thrombi for NETs and fibrin.</p> <h3>Training</h3> <p>The candidate will be trained in Leeds for in vitro methods of clot structure and function, including turbidity, permeation, laser scanning confocal microscopy and scanning electron microscopy. We have a number of research staff (post-doctoral research fellows and research technicians) who will provide the training in these methods. Furthermore, the candidate will be trained in AFM force spectroscopy, magnetic tweezers and turbidimetric analysis of protofibril packing. We have a biophysical post-doctoral research fellow (Dr Stephen Baker) and another PhD student who are well versed with these interdisciplinary methods and will provide training. All these methods have been set up and the expertise is available in house. For the in vivo skills, the candidate will be trained in Leeds, where we have set up our murine thrombosis models using FeCl3 and laser injury of the vessel wall, and in which thrombus formation is analysed by intravital microscopy. For this studentship, we will establish a new model for venous thrombosis that is based on restriction of blood flow in the vena cava by placing ligatures, which leads to the development of venous thrombus over the course of 3-5 days. NETs have been shown to be part of the ensuing thrombus in this model.</p> <h3>Fit with Current On-going Research</h3> <p>Thrombosis is a leading cause of death worldwide and recent studies show an important role for clot structure and function in determining thrombosis risk. Our group focuses on novel mechanisms regulating clot structure and stability with the aim to develop novel diagnostics and therapeutics (BHF programme led by Ari&euml;ns). This studentship will cement existing interactions and create a new link between in vivo studies (Ari&euml;ns, Philippou) and molecular physics (Connell). This studentship will combine the area of expertise in haemostasis and thrombosis (Ari&euml;ns, Philippou) with that in Nanoscale biophysics (Connell).</p> <h3>Fit with Overall Cardiovascular Research Strategy</h3> <p>Cardiovascular disease and thrombosis are one of the major causes of disability and mortality in the ageing population worldwide. Older people are particularly at risk of developing stroke, heart disease or venous thrombosis. However, with increases of the knowledge of the disease processes and advances in surgical procedures, repair and recovery from cardiovascular disease is improving. This research project is focused on elucidating the mechanisms that link inflammation with thrombosis and that determine the structure and function of the blood clot or thrombus. This knowledge and expertise will lead to advances in the early diagnosis and treatment of cardiovascular disease, and improve repair from this devastating disease. Treatments for thrombosis are available but have problems. There is a high risk of bleeding in patients on anticoagulation, even with the newer direct oral anticoagulant inhibitors. Furthermore, treatment of ischaemic stroke with tPA to lyse the fibrin clot is effective only in a proportion of the patients, for reasons that are unknown. A better understanding of the structure of the blood clot and the mechanisms that link inflammation with thrombosis may indicate new direct ways to treat thrombosis, increase efficacy and reduce unwanted side effects such as bleeding.</p> <h3>References</h3> <p>Fuchs TA, Brill A, Duerschmied D, Schatzberg D, Monestier M, Myers DD Jr, Wrobleski SK, Wakefield TW, Hartwig JH, Wagner DD. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci U S A. 2010 Sep 7;107(36):15880-5.</p> <p>Longstaff C, Varj&uacute; I, S&oacute;tonyi P, Szab&oacute; L, Krumrey M, Hoell A, B&oacute;ta A, Varga Z, Komorowicz E, Kolev K. Mechanical stability and fibrinolytic resistance of clots containing fibrin, DNA, and histones. J Biol Chem. 2013 Mar 8;288(10):6946-56.</p> <p>Brill A, Fuchs TA, Savchenko AS, Thomas GM, Martinod K, De Meyer SF, Bhandari AA, Wagner DD. Neutrophil extracellular traps promote deep vein thrombosis in mice. J Thromb Haemost. 2012 Jan;10(1):136-44.</p> <p>Fuchs TA, Brill A, Wagner DD. Neutrophil extracellular trap (NET) impact on deep vein thrombosis. Arterioscler Thromb Vasc Biol. 2012 Aug;32(8):1777-83.</p> <p>Carvalho FA1, Connell S, Miltenberger-Miltenyi G, Pereira SV, Tavares A, Ari&euml;ns RA, Santos NC. Atomic force microscopy-based molecular recognition of a fibrinogen receptor on human erythrocytes. ACS Nano. 2010 Aug 24;4(8):4609-20.</p> <p>Domingues MM, Macrae FL, Duval C, McPherson HR, Bridge KI, Ajjan RA, Ridger VC, Connell SD, Philippou H, Ari&euml;ns RA. Thrombin and fibrinogen &gamma;&lsquo; impact clot structure by marked effects on intrafibrillar structure and protofibril packing. Blood. 2016 Jan 28;127(4):487-95.</p> <p>Allan P, Uitte de Willige S, Abou-Saleh RH, Connell SD, Ari&euml;ns RA. Evidence that fibrinogen &gamma;&lsquo; directly interferes with protofibril growth: implications for fibrin structure and clot stiffness. J Thromb Haemost. 2012 Jun;10(6):1072-80.</p> <p>Duval C, Ali M, Chaudhry WW, Ridger VC, Ari&euml;ns RA, Philippou H. Factor XIII A-subunit V34L variant affects thrombus cross-linking in a murine model of thrombosis. Arterioscler Thromb Vasc Biol. 2016 Feb;36(2):308-16.</p> <p>Brill A, Fuchs TA, Savchenko AS, Thomas GM, Martinod K, De Meyer SF, Bhandari AA, Wagner DD. Neutrophil extracellular traps promote deep vein thrombosis in mice. J Thromb Haemost. 2012 Jan;10(1):136-44.</p>

<p>Please note these are not standalone projects and applicants must apply to the PhD academy directly.</p> <p>Applications can be made at any time. To apply for this project applicants should complete a<a href="https://medicinehealth.leeds.ac.uk/downloads/download/129/faculty_graduate_school_-_application_form"> Faculty Application Form</a> and send this alongside a full academic CV, degree transcripts (or marks so far if still studying) and degree certificates to the Faculty Graduate School <a href="mailto:fmhpgradmissions@leeds.ac.uk">fmhpgradmissions@leeds.ac.uk</a>.</p> <p>We also require 2 academic references to support your application. Please ask your referees to send these <a href="https://medicinehealth.leeds.ac.uk/downloads/download/130/faculty_graduate_school_-_scholarship_reference_form">references</a> on your behalf, directly to <a href="mailto:fmhpgradmissions@leeds.ac.uk">fmhpgradmissions@leeds.ac.uk</a></p> <p>If you have already applied for other projects using the Faculty Application Form this academic session you do not need to complete this form again. Instead you should email fmhgrad to inform us you would like to be considered for this project.</p> <p>If English is not your first language, you must provide evidence that you meet the University&#39;s minimum English language requirements (below).</p>

A degree in biological sciences, dentistry, medicine, midwifery, nursing, psychology or a good honours degree in a subject relevant to the research topic. A Masters degree in a relevant subject may also be required in some areas of the Faculty. For entry requirements for all other research degrees we offer, please contact us.

Applicants whose first language is not English must provide evidence that their English language is sufficient to meet the specific demands of their study. The Faculty of Medicine and Health minimum requirements in IELTS and TOEFL tests for PhD, MSc, MPhil, MD are: &acirc;&euro;&cent; British Council IELTS - score of 7.0 overall, with no element less than 6.5 &acirc;&euro;&cent; TOEFL iBT - overall score of 100 with the listening and reading element no less than 22, writing element no less than 23 and the speaking element no less than 24.

<p>For further information please contact the Graduate School Office<br /> e:<a href="mailto:fmhpgradmissions@leeds.ac.uk">fmhpgradmissions@leeds.ac.uk</a>, t: +44 (0)113 343 8221.</p>