ABCC3基因表达及其mRNA剪接变异对氯吡格雷抵抗的影响

Clopidogrel resistance is a frequently encountered phenomenon in clinical settings. Accumulating evidence has demonstrated that the less formation of clopidogrel active metabolite in the liver is responsible for clopidogrel resistance, and that multidrug resistance-associated protein 3 (also known as MRP3) expressed in the liver mediates the export of some drugs from the hepatocytes to blood circulation, suggesting that a higher mRNA expression level of ABCC3 (which encodes MRP3) may be associated with the less generation of clopidogrel active metabolite in the liver. Our recent work indicated that the formation of clopidogrel active metabolite was less in the ABCC3+/+ mice than in the age- and gender-matched ABCC3-/- mice when the same dose of clopidogrel was given orally, with the former having a lower antiplatelet effect. We hypothesized that the higher expression levels of ABCC3 mRNA could play an important role in the less formation of clopidogrel active metabolite, and consequently clopidogrel resistance. It has been known that there are up to 17 alternative splicing variants of ABCC3 mRNA in human genome, resulting in marked polymorphisms in MRP3 protein structure and function, suggesting clopidogrel resistance in relation to increased ABCC3 mRNA expression levels or to greater portion of loss-of-function ABCC3 mRNA alternative splicing variant transcripts in total ABCC3 mRNA transcripts. This work was designed to classify all recruited healthy subjects and patients with coronary artery disease into four quartiles (Q1 - Q4), respectively, according to the descending order of inhibition of ADP-induced platelet aggregation before and after dosing of 300 mg clopidogrel. Clopidogrel-sensitive (Q1) versus clopidogrel-resistant (Q4) groups were chosen to compare between-group differences in the levels of ABCC3 mRNA expression, relative expression levels of each ABCC3 mRNA splicing variant of interest, and the plasma concentrations of clopidogrel active metabolite, respectively. This work would be used to clarify the potential relationship between polymorphic profiling of ABCC3 mRNA expression and the formation of clopidogrel active metabolite as well as clopidogrel resistance. These efforts would provide pharmacogenetic basis of personalized medicine of clopidogrel in patient care.

氯吡格雷抵抗的主要原因是肝脏生成的氯吡格雷活性产物减少。相关研究表明，外周血白细胞ABCC3/MRP3 mRNA表达增加与氯吡格雷抵抗有关，但其确切机制尚不清楚。我们的前期研究显示，ABCC3+/+小鼠生成的氯吡格雷活性产物较ABCC3-/-小鼠显著减少，进而产生较弱的抗血小板作用。已知人mRNA可变剪接可显著改变其表达与功能。据此推测人ABCC3 mRNA表达及其可变剪接可显著改变MRP3表达水平和肝细胞内氯吡格雷活性产物的生成量，进而参与氯吡格雷抵抗形成。本课题将系统研究多种ABCC3 mRNA剪接体在稳定转染细胞株的表达与功能改变，并在健康受试人群中研究这些剪接体与氯吡格雷活性产物血浓度和氯吡格雷抗血小板作用的相关性及其机制，明确ABCC3 mRNA可变剪接体与氯吡格雷抵抗在冠心病病人中的相互关系。此项目旨在阐明ABCC3 mRNA表达及其可变剪接参与氯吡格雷抵抗形成的新机制。

氯吡格雷是一种最常用的抗血小板药物。氯吡格雷本身是一种无活性的前药，通过在肝脏中被代谢活化（转化）后生成其活性代谢产物H4来阻断体内ADP对血小板ADP受体P2Y12的结合与激活而抑制ADP诱导的血小板聚集作用。在临床上，约有15% – 45%的冠心病病人在口服氯吡格雷后不能获得其预期的抗血小板疗效。目前已知的部分机制是肝脏药物代谢酶CYP2C19的基因多态性及其活性的个体差异。因此，进一步深入研究影响氯吡格雷疗效的其它未知因素或机制具有重要的科学意义和临床应用前景。.本项目的主要研究结果显示：与野生型（WT）小鼠比，Abcc3 基因敲除（KO）小鼠表现为氯吡格雷的代谢活化能力增强，因此其抗血小板作用也增强，与预期的研究结果相吻合。在后续相关研究中，鉴定了人UGT2B7是催化氯吡格雷葡萄糖醛酸结合物（CLP-G）生成的主要II相代谢酶；首次发现人MRP3（ABCC3基因编码的转运体）是将肝细胞内生成的CLP-G转运（外排）到血液的转运体。在细胞水平，发现人ABCC3 mRNA的剪切变异体ABCC3-013的外排转运能力较对照ABCC3-002显著增强；在口服氯吡格雷的冠心病病人中，外周血白细胞ABCC3-013的mRNA水平在氯吡格雷抵抗组显著高于非抵抗组，通过构建ROC曲线评估了外周血细胞的ABCC3-013 mRNA水平对氯吡格雷抵抗的潜在诊断价值，其诊断灵敏度为52%，特异性为86%，相关结果已分别申请国家发明专利、PCT和美国专利各1项。深入探索氯吡格雷抵抗的新机制，首次发现人补体抑制剂C4BP-a蛋白血清水平在氯吡格雷抵抗组显著低于非抵抗组，可作为预测氯吡格雷抵抗的生物标志物，现申请国家发明专利1项。新发现氯吡格雷可显著诱导线粒体mARC1表达，在氯吡格雷抵抗组显著低于非抵抗组。相关工作在被完善后将申报国家发明专利1项。.相关系列研究工作发现了与“氯吡格雷抵抗”有关的3种新机制，对系统阐明氯吡格雷抵抗的形成机制有一定的理论意义。除已发表的6篇相关SCI论文外，新发现均已申请了国家发明专利以期进一步开发其相应的临床检测试剂盒，通过科技创新成果的不断转化以满足临床的实际需求。