金属蛋白酶ADAMTS-13对von Willebrand factor还原作用的机制研究

Coronary artery disease (CAD) is the leading cause of death around the world. The thrombi formed within the vessels block the blood flow of coronary arteries, which lead to myocardial infarction. von Willebrand factor (VWF) multimers are critical for thrombus formation through recruiting and aggregating platelets in plasma. The size and function of VWF multimers can be regulated by the metalloprotease ADAMTS-13 to maintain the normal hemostasis in the body. Normal VWF multimers circulating in plasma do not bind platelets, whereas they are activated by the high shear stress to bind and aggregate platelet spontaneously, resulting in the bleeding tendency because of the consumption. The previous studies have shown that plasma VWF multimers were induced by the shear stress to form the disulfide bonds through surface-exposed free thiols. Recently, there were several reports that the reductants can regulate the size and function of VWF multimers by reducing the disulfide bonds and the effects are reversible. Our study has further demonstrated that ADAMTS-13 contains a disulfide bond reducing activity that primarily targets disulfide bonds in plasma VWF multimers induced by high shear stress. Interestingly, ADAMTS-13 acts as a reductase by using the cysteine thiols in its non-proteolytic regions that remain exposed after being subjected to hydrodynamic forces. Blocking these active thiols eliminates the reducing activity and moderately decreases ADAMTS-13 activity in cleaving ULVWF strings under flow conditions. The present study will further investigate which cysteine thiols in ADAMTS-13 play key roles to reduce VWF multimers and how shear stress affects the process. Since VWF multimers are important in developing the CAD lesions, we will measure the thiol/disulfide pattern of VWF multimers in the plasma of CAD patients and associate with the severity of the disease.

冠状动脉心脏病（冠心病）是导致人类死亡的首要因素。在血管中形成的血栓如果堵塞冠状动脉，将造成心肌梗死的发生。血浆中的von Willebrand factor (VWF) 多聚体可聚集活化血小板，在血栓形成过程中至关重要。血浆中金属蛋白酶ADAMTS-13通过剪切VWF多聚体，调节机体凝血与血栓系统的功能。在高速流体剪切力作用下，正常VWF多聚体通过表面游离巯基重组形成新的二硫键，其活性部位暴露后与血小板主动结合形成血小板血栓，导致高活性的VWF多聚体的消耗，从而使患者的出血倾向增高。本课题组进一步证实，ADAMTS-13阻止流体剪切力导致的VWF二硫键置换，其还原活性是通过非蛋白酶结构域内的活性游离巯基起作用。本申请将研究对ADAMTS-13还原活性起关键作用的活性游离巯基，以及流体剪切力对此调节功能的影响。同时检测冠心病患者中VWF多聚体二硫键分布的变化，探讨其与疾病发生发展的关联。

冠状动脉心脏病（冠心病）是导致人类死亡的首要因素。在血管中形成的血栓如果堵塞冠状动脉，将造成心肌梗死的发生。血浆中的von Willebrand factor (VWF) 多聚体可聚集活化血小板，在血栓形成过程中至关重要。血浆中金属蛋白酶ADAMTS-13通过剪切VWF多聚体，调节机体凝血与血栓系统的功能。在高速流体剪切力作用下，正常VWF多聚体通过表面游离巯基重组形成新的二硫键，其活性部位暴露后与血小板主动结合形成血小板血栓，导致高活性的VWF多聚体的消耗，从而使患者的出血倾向增高。本课题组进一步证实，ADAMTS-13阻止流体剪切力导致的VWF二硫键置换，其还原活性是通过非蛋白酶结构域内的活性游离巯基起作用。本申请将研究对ADAMTS-13还原活性起关键作用的活性游离巯基，以及流体剪切力对此调节功能的影响。同时检测冠心病患者中VWF多聚体二硫键分布的变化，探讨其与疾病发生发展的关联。.本研究对患者年龄、性别与炎性状态、血凝、以及血脂指标的相关性进行分析；探究对ADAMTS-13还原活性起关键作用的活性游离巯基，以及流体剪切力对此调节功能的影响；利用VWF瑞斯托霉素辅因子活性检测（vWF:Rcof）的方法分别对冠心病患者血浆（175例）和瓣膜病患者的血浆（180例）进行了VWF的抗原检测，结果显示，在冠心病患者中的VWF水平明显高于瓣膜病患者。