Role of NETs in thrombolytic resistance of ischaemic stroke thrombi: a novel quantitative approach

Acute ischaemic stroke is a major cause of death and disability in the UK. It occurs if a blood vessel within the head becomes blocked, leading to loss of blood supply and damage to a part of the brain. The blockage is usually made up of thrombus or blood clot, which may have formed where the blood vessel is already narrowed by fatty deposits which develop as people age. Sometimes, the thrombus may instead have formed within the heart or large blood vessels soon after they leave the heart, with the thrombus then being carried in the blood stream until reaching and blocking a smaller diameter blood vessel within the head.

If a stroke patient reaches hospital in less than three to four hours after the beginning of the stroke, current standard treatment is to attempt to unblock the artery using a drug (known as rt-PA). This drug was designed to break down fibrin, a major protein component of thrombus. Unfortunately, clinical trials have shown that on average this drug only produces quite small benefits. This is probably because the drug fails to break down the thrombus and unblock the blood vessel in a significant proportion of patients treated. Possible reasons for this failure include the fact that thrombus contains other components, other than fibrin. Recently published work has identified the presence of neutrophil extracellular traps (NETs) within stroke-causing thrombus. NETs consist of fibres of genetic material, DNA, which have been released by certain types of white blood cell (neutrophils) in the blood stream. The structure of NETs is very different from that of fibrin, and therefore rt-PA may be incapable of breaking down NETs. Hence, if stroke-causing thrombus contains a significant amount of NETs, this may explain the failure of rt-PA sometimes to break down thrombus.

In this project, we seek to answer the question of how the quantity of NETs in stroke-causing thrombus changes the effectiveness of rt-PA in breaking down thrombus. Furthermore, we seek to answer the question of whether more effective thrombus breakdown is achieved when another drug, which is designed to break down DNA (DNA being the major component of NETs), is used in addition to rt-PA, and whether the additional effectiveness is related to the quantity of NETs.

In this project, we explore these questions using stroke-causing thrombus which has been recovered from patients undergoing another established treatment for stroke, known as mechanical thrombectomy; this is a surgical procedure in which a device is inserted into the patient's blocked blood vessel and the blockage is sucked or pulled out. We will measure the quantity of NETs in each patient thrombus by a novel method which involves separating the thrombus into individual cells and then counting the white blood cells which are producing NETs. The breakdown of the thrombus will be measured by weighing it after the drugs are applied to the thrombus in a "test tube", as the amount of thrombus which remains will decrease and weigh less as the thrombus is broken down. Thus, we will be able to see if there is a relationship between the amount of NETs in each thrombus and how well it is broken down by the drugs.

It is our expectation that the thrombus most susceptible to break down with rt-PA is the thrombus which contains the least NETs, and that combined treatment with rt-PA and a DNA-targeting drug is more effective at achieving thrombus break down than rt-PA alone. If our experiments prove these ideas, then this will demonstrate the importance of NETs as a factor impairing successful thrombus break down, and will demonstrate the importance of including a DNA-targeting drug as part of the treatment for stroke. This could pave the way towards a human clinical trial to properly compare the efficacy of using a combination treatment of rt-PA and a DNA-targeting drug against the efficacy of using the standard current treatment of rt-PA alone.

BACKGROUND: Recombinant tissue plasminogen activator (rt-PA), the current licensed thrombolytic agent used in stroke treatment, often fails to reopen large diameter arteries occluded by thrombus. Recent experiments in retrieved stroke thrombi have demonstrated the presence of neutrophil extracellular traps (NETs). Additionally, in ex-vivo experiments, DNAse and rt-PA together resulted in faster thrombolysis than rt-PA alone.
AIM: To apply a novel method of NETs quantitation to formally probe the role of NETs in a) thrombolytic resistance to rt-PA, b) enhanced thrombus digestion with combined DNAse and rt-PA treatment ex-vivo, c) the size of the occluding thrombus in-vivo.
METHODS: The concentration of neutrophils and the quantity of appendant NETs will be measured by flow cytometry in mechanical thrombectomy specimens from stroke patients, after thrombus dissociation into single cellular elements by use of accutase. The relationship between the quantity of NETs and the effect of combinations of DNase and rt-PA on thrombolysis ex-vivo will be studied. The degradation of NETs will be examined in the thrombus breakdown products.
VALUE: Understanding the contribution of NETs to thrombus stability is a first and essential step in developing thrombolytic approaches which target NETs, with the ultimate goal of improving clinical outcomes in stroke.