Analysis of shear stress-induced von Willebrand factor and platelet damage from ventricular assist devices

心室辅助装置（VAD）支持心力衰竭患者的心脏功能。VAD将血液从心脏泵送到身体的各个部位。虽然心力衰竭可以用VAD治疗，但出血仍然是长期使用VAD的严重并发症。获得性血管性血友病（aVWD）是由于高分子量（HMW）血管性血友病因子（vWF）多聚体的分解。血液中循环的vWF在高剪切应力下被ADAMTS 13金属蛋白酶切割。HMW vWF多聚体是血小板聚集所必需的，这对血液凝固至关重要。本研究将研究剪切应力下血液中HMW vWF的损失。因此，本项目的假设是ADAMTS 13的抑制可以恢复HMW vWF多聚体和vWF功能。这一假设将通过使用以下三个目标进行测试：1。分析ADAMTS 13抑制对血液中HMW vWF多聚体水平和活性的影响。将在存在或不存在ADAMTS 13抑制剂的情况下分析HMW多聚体的损失和暴露于剪切应力的vWF活性。将通过免疫印迹和血小板聚集、流式细胞术和共聚焦显微镜分析血液。2.研究通过体外VAD或心肺转流（CPB）回路的血液中HMW vWF多聚体的水平和活性。将通过免疫印迹和血小板聚集来评估旁路手术期间通过VAD的健康志愿者或通过CPB回路的心脏病患者的血液中的vWF HMW多聚体和活性。这是为了确定机械剪切应力是否对健康志愿者和心力衰竭患者的血液有不同的影响。由于不可能获得2000 mL的血液，这需要在体外泵送通过VAD，我们将在本研究中使用旁路手术期间获得的血液。分析植入VAD的患者和绵羊血浆中vWF HMW多聚体的水平和活性。从植入VAD的绵羊（Calon Cardio Technology Ltd/Catholic Univ. of Leuven）和人类患者（来自HDZ-NRW）获得的血浆样本将用于评估vWF的HMW多聚体水平（通过免疫印迹法）和活性（通过血小板聚集法）。这种分析将有助于研究剪切应力诱导的ADAMTS 13 HMV vWF多聚体损失在真实的生理conditions.Significance的影响：心力衰竭是英国最大的杀手。由于心脏移植供体的可用性下降，VAD植入已成为移植或目的地治疗的桥梁。因此，了解长期使用VAD对患者生活质量的影响变得至关重要。获得性von Willebrand综合征是一种出血性疾病，在VAD植入患者中很常见。因此，拟议的研究将有助于开发低血液损伤的VAD。拟议的项目属于EPSRC研究主题的临床器械。该研究将有利于设计副作用较少的VAD，突出了医疗保健应用的翻译。该项目将有利于新的医疗保健解决方案的开发，这些解决方案属于开发未来疗法的主题的重大挑战。

A ventricular assist device (VAD) supports heart function in people with heart failure. VADs pumps blood from the heart to various parts of the body. Although heart failure can be treated with VADs, bleeding remains a serious complication of long-term VAD use. Acquired von Willebrand Disease (aVWD) is due to the breakdown of high molecular weight (HMW) von Willebrand factor (vWF) multimers. vWF circulating in blood is cleaved by ADAMTS13 metalloprotease under high shear stress. HMW vWF multimers are required for platelet aggregation, which is critical for blood clotting. This study will investigate the loss of HMW vWF from blood under shear stress. Therefore, the hypothesis of this project is that inhibition of ADAMTS13 can restore HMW vWF multimers and vWF function. This hypothesis will be tested by using the following three aims:1. To analyse the effect of ADAMTS13 inhibition on the levels and activity of HMW vWF multimers in blood. The loss of HMW multimers and activity of vWF exposed to shear stress will be analysed in the presence or absence of ADAMTS13 inhibitors. Blood will be analysed by immunoblotting and platelet aggregation, flow cytometry and confocal microscopy. 2. To study the levels and activity of HMW vWF multimers in blood passed through a VAD in vitro or by cardiopulmonary bypass (CPB) circuit. The vWF HMW multimers and activity in plasma of blood of healthy volunteers passed through a VAD or heart patients passed through CPB circuit during bypass surgery will be assessed by immunoblotting and platelet aggregation. This is to determine if mechanical shear stress differentially affects blood from healthy volunteers and heart failure patients. Since it is impossible to obtain 2000 mL of blood, which is required to pump through a VAD in vitro, we will be using blood obtained during bypass surgery in this study.3. To analyse the levels of vWF HMW multimers and activity in plasma of patients and sheep implanted with a VAD. The plasma samples obtained from a VAD implanted sheep (Calon Cardio Technology Ltd/Catholic Univ. of Leuven) and human patients (from HDZ-NRW) will be used to assess the HMW multimer levels (by immunoblotting) and the activity (by platelet aggregation) of vWF. This analysis will be useful to study the effect of shear stress-induced ADAMTS13 HMV vWF multimers loss in real physiological conditions.Significance: Heart failure is the UK's biggest killer. Since the availability of heart donors for transplantation has declined, VAD implantation has become an option for bridge to transplantation or destination therapy. Therefore, it becomes pertinent to understand the consequences of long-term VAD usage on the quality of life of patients. Acquired von Willebrand syndrome, a bleeding disorder, is common in VAD implanted patients. The proposed research will, therefore, be useful in the development of lower blood damage VADs.The proposed project is within Clinical Devices of EPSRC research topic. The research will be beneficial in designing VADs with less side-effects highlighting the translation to healthcare applications. The project will advantage the development of new healthcare solutions that comes under the Developing Future Therapies grand challenge of the theme.