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

急性缺血性卒中是英国死亡和残疾的主要原因。它发生在头部血管阻塞，导致血液供应丧失和大脑部分受损的情况下。阻塞通常由血栓或血块组成，这些血栓或血块可能是在血管已经因随着年龄增长而形成的脂肪沉积而变窄的地方形成的。有时，血栓可能在离开心脏后不久就在心脏或大血管内形成，然后血栓被携带在血流中，直到到达并阻塞头部内直径较小的血管。如果中风患者在中风开始后不到三到四小时内到达医院，目前的标准治疗是尝试使用药物（称为rt-PA）来疏通动脉。这种药物被设计用来分解血栓的主要蛋白质成分纤维蛋白。不幸的是，临床试验表明，平均而言，这种药物只产生相当小的好处。这可能是因为在相当一部分接受治疗的患者中，药物未能分解血栓和疏通血管。这种失败的可能原因包括血栓含有除纤维蛋白以外的其他成分。最近发表的工作已经确定了中性粒细胞胞外陷阱（NET）的存在下，中风引起的血栓。NET由遗传物质DNA纤维组成，DNA是血液中某些类型的白色血细胞（中性粒细胞）释放的。NET的结构与纤维蛋白的结构非常不同，因此rt-PA可能无法分解NET。因此，如果中风引起的血栓中含有大量的NETs，这可能解释rt-PA有时不能分解血栓的原因。在本项目中，我们试图回答中风引起的血栓中NETs的数量如何改变rt-PA分解血栓的有效性的问题。此外，我们试图回答这样一个问题，即当另一种旨在分解DNA的药物被用于治疗血栓时，（脱氧核糖核酸是母语英语教师的主要成分），是否与rt-PA配合使用，以及额外的成效是否与母语英语教师的人数有关。在这项计划中，我们使用导致中风的血栓来探讨这些问题，这些血栓是从接受另一种已知的中风治疗（称为机械血栓切除术）的患者中恢复的;这是一种外科手术，将一个装置插入病人阻塞的血管中，然后将阻塞物吸出或拔出。我们将通过一种新的方法测量每个患者血栓中NET的数量，该方法包括将血栓分离成单个细胞，然后对产生NET的白色血细胞进行计数。将药物应用于“试管”中的血栓后，通过称重来测量血栓的分解，因为随着血栓分解，剩余的血栓量将减少并且重量更轻。因此，我们将能够看到每个血栓中NETs的量与药物对其的分解程度之间是否存在关系。我们预计，最容易被rt-PA分解的血栓是含有最少NETs的血栓，并且rt-PA和DNA靶向药物的联合治疗比单独使用rt-PA更有效地实现血栓分解。如果我们的实验证明了这些想法，那么这将证明NET作为阻碍成功血栓分解的因素的重要性，并将证明将DNA靶向药物作为中风治疗的一部分的重要性。这可以为人类临床试验铺平道路，以适当地比较使用rt-PA和DNA靶向药物的联合治疗与使用rt-PA单独的标准当前治疗的疗效。

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.