阿司匹林抗血小板反应多样性在卒中治疗中的意义及机制研究

Aspirin as an anti-platelet drug is known to have variability of platelet response, which is manifested by the existence of aspirin resistance in some patients (reportedly in more than 20% of the patients). And it has been indicated that individualized aspirin therapy is urgently needed in clinic practice. Previously our team has found that up to 20% of the stroke patients also have AR, which is an independent factor of ischemic event; meanwhile, gene polymorphism may be one of the most important factors responsible for AR. To date, multiple gene single nucleotide polymorphisms, such as GP, P2RY, ITGA,PTGS, TBXA, and PEAR have been shown to be correlated with AR in the field of cardiovascular, but the relevance differs in different samples, groups and races. There have been few researches in the field are related to stroke, and the relationship between genetic polymorphisms, AR and ischemic events is not fully understood yet. This prospective clinical research plans to explore the relationship between aspirin pharmacogenomics, platelet function and clinical ischemic events. The up-to-date platelet aggregation array VerifyNow system and proteomic profiling will be used for patients with acute ischemic stroke, to understand the relationship between related genetic polymorphisms and AR and to establish a genetic map for stroke patients with AR in China. Moreover, we will also study the influence of aspirin at both standard and increased doses on AR and recurrence of ischemic events, and to explore the possible mechanism, hoping to lay a foundation for accurate and individualized aspirin treatment of stroke patients.

目前已知抗血小板药物阿司匹林具有血小板反应多样性，表现为部分患者存在阿司匹林抵抗（AR）。在心血管领域，已发现GP、ITGA、P2RY 、PTGS、TBXA、PEAR等基因多态性（SNP）与AR相关，但其关联性在不同样本、群体、种族间的结果不一致，且在卒中领域同时经血小板功能检测和缺血事件再发验证的鲜有报道。申请人前期研究发现中国卒中人群AR的发生率高达20%，是缺血性事件再发的独立危险因素，其中SNP是AR最重要影响因素之一。拟在本项目中，利用国际公认的血小板聚集检测仪VerifyNow在卒中患者中明确是否存在AR，增加阿司匹林剂量是否对AR有影响，再使用PCR结合测序技术在AR患者中检测其SNP，通过随访缺血事件的再发，从而明确特异性SNP与再发缺血事件的关联。此外，还将初步探索特异性SNP影响血小板聚集的分子机制。这一项目，有望为精准医疗下的阿司匹林个体化抗血小板治疗奠定循证医学基础

抗血小板药物阿司匹林具有血小板反应多样性，表现为部分患者存在阿司匹林抵抗（AR）。在心血管领域，已发现基因多态性与AR相关，但其关联性在不同样本、群体、种族间的结果不一致，且在卒中领域同时经血小板功能检测和缺血事件再发验证的鲜有报道。本课题在1008例新发缺血性卒中患者中开展前瞻性研究，利用质谱法（MassArray）共检测已知的可能与AR相关性较强的的13个基因共18个SNP位点，未检测到GP1BA（rs2243093）；利用多重 PCR技术和MassArray 技术发现PCSK9（C>T，rs662145）是影响阿司匹林抗血小板作用的重要基因，在本研究人群中突变基因频率为13.33%。本研究采用国际先进的VerifyNow检测仪检测血小板聚集功能，将ARU≥550定义为AR，该研究人群中表现为AR共202例，约占20.04%。202例AR患者增加阿司匹林剂量至150mg/d，24例（11.9%）转化为阿司匹林相对抵抗（ASR），178例（88.1%）为阿司匹林绝对抵抗（ARR）。共有816例患者完成了一年随访，其中72例患者出现临床终点事件，包括主要终点事件66例，次要终点事件6例。研究发现PCSK9 (C>T, rs662145) 与AR密切相关（p=0.021），但与临床主要终点事件无明显关联性（p=0.076），AR是缺血性事件再发的独立危险因素（HRadj 3.28 [95% CI 1.52-7.71]，p = 0.002）。基础研究发现，随着PCSK9（C>T，rs662145）突变位点的增多，Src、ERK5和JNK磷酸化的表达逐渐增强，提示PCSK9可能是通过激活Src、ERK5和JNK 蛋白磷酸化促进血小板活化，从而减弱阿司匹林抗血小板作用。本课题在中国新发缺血性卒中人群中发现PCSK9（C>T，rs662145）是影响阿司匹林抗血小板作用的重要基因，与AR密切相关，AR是缺血性事件再发的独立危险因素，增加阿司匹林剂量可部分改善AR。本课题初步建立阿司匹林临床疗效评价体系，有效指导临床抗血小板药物的选择以及阿司匹林剂量的调整，为实现个体化抗血小板治疗的精准医学模式奠定循证医学基础。