基于前期多项临床研究的ADAMTS家族蛋白血栓相关共同结构域发现及其非编码SNP功能的研究

ADAMTS family proteins are newly discovered multi-domain protein hydrolase that contains platelet-binding protein. This is an important progress in thrombosis research in recent years. Some of the ADAMTS family proteins are involved in thrombosis and vascular diseases, but their mechanism of action is far from elucidated. Results of multiple anti-thrombotic drug use precision study initiated by different applicants showed that anti-thrombotic efficacy-related gene polymorphisms are densely distributed in the ADAMTS gene. We speculate that ADAMTS family proteins may have played an important role in thrombosis and individual antithrombotic differences. They may be potential new biomarkers and pharmacological targets. In this research project, we will use bioinformatics and computer simulation to analyze the where the SNPs found in the early stage are located on ADAMTS DNA, and speculate the functions of these SNPs. We will predict the common thrombus-associated domains of ADAMTS according to the characteristics of exon SNPs, design the antibodies, and then construct genetically modified mouse model for further validation. For non-coding SNPs, we intend to use CRISPR / Cas9 gene to edit pluripotent stem cells , and then induce them into platelets to explore the role and mechanism of the SNPs. The purpose of this study was to reveal the effect of ADAMTS family proteins on thrombosis and anti-thrombotic efficacy and its mechanism, and to explore and establish new methods for non-coding SNPs function study.

ADAMTS家族蛋白是新发现的含有血小板结合蛋白的多结构域蛋白水解酶，是近年来血栓研究的重要进展，但其影响血栓及血管性疾病的作用机理还远未阐明。申请人发起的多个抗血栓药物临床研究结果显示，与抗血栓药效相关的基因多态性在ADAMTS基因上密集分布，我们推测ADAMTS家族蛋白可能在血栓形成和抗血栓药效个体差异中发挥着重要作用，是潜在的生物标志物和药理学新靶点。在本课题中，我们将用生物信息学和计算机模拟分析前期发现的SNP所在ADAMTS DNA的位置并推测其功能，根据外显子SNP的特点预测ADAMTS与血栓相关的共同结构域，然后设计抗体、构建基因修饰小鼠模型验证；对非编码SNP用CRISPR/Cas9基因编辑多能干细胞，然后诱导成血小板探索该SNP的作用及机理。本研究旨在揭示ADAMTS家族蛋白对血栓形成及对抗血栓药效的影响及作用机理，并探索建立非编码SNP功能研究的新方法。

ADAMTS家族蛋白是新发现的含有血小板结合蛋白的多结构域蛋白水解酶，是近年血栓研究的重要进展，多个家族蛋白参与了血栓及血管性疾病，但它们的作用机理还远未阐明。本研究通过对4个抗血栓药物6个队列研究数据进行分析，验证ADAMTS基因的SNP对抗血栓药物治疗药效和临床事件存在显著影响。研究发现ADAMTS13、ADAMTS9、ADAMTS19等可能在血栓形成中发挥了重要作用。采用CRISPR/Cas9基因编辑方法构建ADAMTS13基因敲除小鼠模型，iPS技术构建ADAMTS13表达调控的内皮细胞，研究ADAMTS13基因敲除对小鼠肺栓塞模型中血栓形成影响因素，体外、小鼠体内给予PF4蛋白的干预，评价血小板计数、血小板凋亡、血小板激活受体表达，病理切片和蛋白表达差异磷酸化实验评价血栓形成与蛋白表达差异，进行流式细胞术来测定血小板特异性表面分子（CD61,CD62P）评估血小板激活情况。研究发现ADAMTS13蛋白低表达增加血栓的形成，ADAMTS13通过PF4-ADAMTS13-vWF轴调控血栓和止血，并在血小板减少症中有重要的作用。