CYP2C19基因多态性影响稳定型冠心病患者氯吡格雷治疗后血小板P2Y12受体信号通路的分子机制

徐化洁 赵 昕 吴鸿谊 樊 冰

(复旦大学附属中山医院心内科 上海 200032)

CYP2C19基因多态性影响稳定型冠心病患者氯吡格雷治疗后血小板P2Y12受体信号通路的分子机制

徐化洁 赵 昕 吴鸿谊 樊 冰△

(复旦大学附属中山医院心内科 上海 200032)

目的 探讨中国冠心病患者CYP2C19基因多态性影响氯吡格雷疗效的分子机制。方法 入组208例接受冠状动脉介入治疗(percutaneous coronary intervention,PCI)的稳定型冠心病(stable coronary artery disease,SCAD)患者,采用PCR产物直接测序的方法对所有纳入对象进行CYP2C19基因多态性分析,血栓弹力图检测300 mg氯吡格雷负荷量24 h后的血小板聚集率,流式细胞仪检测P2Y12受体下游信号分子血管扩张刺激磷蛋白血小板反应性指数(vasodilator-stimulated phosphorylation-platelet reactivity index,VASP-PRI)和Akt磷酸化(P-Akt)水平。结果 208例患者中85例(40.9%)的基因型为CYP2C19*1/*1 (非携带者),无等位基因缺失(loss-of-function,LOF),93例(44.7%)为CYP2C19*1/*2或CYP2C19*1/*3(1个LOF),30例(14.4%)为CYP2C19*2/*2或CYP2C19*2/*3 (2个LOF)。氯吡格雷治疗后的血小板聚集率及VASP-PRI随着携带LOF数目的增加而增加,而P-Akt只在2个LOF的情况下才会明显增加。VASP-PRI与氯吡格雷治疗后血小板聚集率有相关性(r=0.672,P<0.001),但是与P-Akt不相关。结论 携带2个CYP2C19 LOF的SCAD患者经氯吡格雷治疗后血小板聚集率更高,并与活性更高的G蛋白偶联受体P2Y12信号通路相关。

稳定型冠心病;CYP2C19; 氯吡格雷; P2Y12; VASP; Akt; 血小板反应性

氯吡格雷作为第二代噻吩吡啶类药物和选择性的血小板P2Y12受体抑制剂,联合阿司匹林可降低冠心病患者经皮冠状动脉介入(percutaneous coronary intervention,PCI)术后的心血管事件风险[1-2]。氯吡格雷是一种无活性的前体药物,需要经过细胞色素P450 (cytochrome P450,CYP450)代谢成活性形式,从而阻止血小板的聚集[3],这个过程中CYP2C19基因起到重要的作用。多项研究显示CYP2C19基因多态性会导致CYP450功能下降,因而减少氯吡格雷活性形式的转化,导致氯吡格雷抗血小板反应性的疗效降低[4-5]。CYP2C19基因多态性主要有CYP2C19*2和*3等位基因缺失(loss-of-function,LOF),在不同的人群中均被证实与血小板高反应性及临床心血管不良事件有着一定的关系[6]。我们之前的研究显示超过60.6%的中国冠心病患者携带CYP2C19 LOF,而只有携带2个CYP2C19 LOF(慢代谢型)的患者临床预后不良[7-8]。然而,国内外的研究聚焦于CYP2C19 LOF与临床事件的关系,并没有深入探讨其对血小板反应性的分子机制的影响。

血小板P2Y12受体偶联于Gi蛋白,ADP激活P2Y12后使得Gi蛋白释放αi和βγ亚基,导致血小板聚集状态的放大及稳定[9]。αi亚基抑制腺苷酸环化酶(adenylyl cyclase,AC),降低环磷酸腺苷(cyclic adenosine monophosphate,cAMP)的浓度,进一步阻止血管扩张刺激磷蛋白(vasodilator-stimulated phosphorylation,VASP)的磷酸化[10]。βγ亚基通过激活激活磷脂酰肌醇3激酶(phosphatidylinositol 3-kinase,PI3K)使得丝氨酸-苏氨酸蛋白激酶B(serine-threonine protein kinase B,PKB,即Akt)磷酸化水平增高[10]。这一系列血小板内信号分子的变化主要涉及VASP及Akt的磷酸化,构成了血小板P2Y12受体的信号转导通路(图1)。本研究拟探索中国冠心病患者氯吡格雷治疗后CYP2C19基因多态性对血小板内信号通路的影响机制,以进一步了解其导致不良临床事件的分子机制。

ADP:Adenosine diphosphate;AC:Adenylate cyclase;cAMP:Cyclic adenosine monophosphate;ATP:Adenosine triphosphate;PI3K:Phosphatidylinositol 3-kinase;PKA:Creatine phosphokinase.

图1 氯吡格雷特异性拮抗ADP与P2Y12受体结合的信号通路

Fig 1 Singnal pathway of platelet ADP P2Y12receptor inhibited by clopidogrel

资 料 和 方 法

研究对象 选择2015年8月至2016年10月在复旦大学附属中山医院心内科接受PCI治疗的208例稳定性心绞痛患者,患者均接受300 mg氯吡格雷和300 mg阿司匹林负荷量治疗。排除标准:年龄小于18岁或者大于80岁,使用GPⅡb/Ⅲa受体抑制剂,联合西洛他唑治疗,对氯吡格雷或阿司匹林过敏或禁忌,恶性肿瘤,妊娠,严重肾功能不全或肝功能不全,血小板总数小于100×109/L,出血风险较高,血液系统疾病。本研究得到复旦大学附属中山医院医学伦理委员会批准,并获得患者的知情同意书。

血小板聚集率测定 在阿司匹林300 mg和氯吡格雷300 mg负荷后24 h,采用血栓弹力图(thrombelastogram,TEG)(美国Haemoscope Corp公司)测定血小板反应性。空腹采血,分别用枸橼酸钠抗凝和肝素钠抗凝静脉血3 mL。TEG分析仪第1通道加入340 μL高岭土处理过的血样(枸橼酸钠抗凝血标本),该通道反映了血小板和纤维蛋白聚集的最大强度MAThrombin;第2通道加入10 μL激活剂F和360 μL肝素化全血,得到纤维蛋白网强度MAFibrin;依次得到第3通道MAAA和第4通道MAADP。血小板聚集率%=[(MAADP-MAFibrin)/(MAThrombin-MAFibrin)] ×100%

CYP2C19基因多态性测定 取静脉血2 mL,采用基因组DNA提取全血样品QIAGEN试剂盒(德国Qiagen公司),按照使用说明书提取基因组DNA,针对CYP2C19*2 (rs4244285) 和CYP2C19*3 (rs4986893)基因多态性设计上游引物5’-CATCTTTGATTCTCTTGT-3’和5’-CCAATCA-TTTAGCTTCAC-3’,下游引物5’-TGAATCAC-AAATACGCAA-3’和5’-ATATTCATTTCCTG-TGCA-3’。PCR扩增在30μL体系中进行,取DNA 100～200 ng、上下游引物各0.5μL、KOD-Plus-Neo聚合酶体系5.4μL,双蒸水补足体积,扩增反应在PCR仪上以94 ℃预变性5 min,然后94 ℃变性30 s,55 ℃退火30 s,72 ℃延伸45 s,35个循环,最后以72 ℃延伸5 min。取 5 μL产物于2.0% 琼脂糖凝胶电泳(溴化乙锭染色)检测。PCR 产物纯化及测序反应由上海杰李生物公司完成。

流式细胞仪检测 根据VASP检测试剂盒(法国Diagnostica Stago/Biocytex公司)的说明书,分别取10 μL枸橼酸钠抗凝全血加入PGE1及PGE1+ADP(终浓度10μmol/L),室温孵育10 min,10μL固定液固定5 min,终止反应。孵育VASP磷酸化特异性鼠单抗或阴性对照抗体及血小板特异性抗体CD61-PE,最后孵育多克隆抗鼠IgG抗体,使用Coulter Epics XL-M 流式细胞仪(美国Beckman/Coulter公司)检测。VASP-PRI (%)=(MFIPGE1-MFIPGE1+ADP)/MFIPGE1× 100 (%)

流式细胞仪检测P-Akt (Ser473) 取10 μL抗凝全血溶解于300 μL PBS中,室温下加入10 μmol/L ADP静置5 min,再加4%多聚甲醛100 μL固定2.5 h。按照上述试验步骤,分别孵育Akt Ser473-FITC及CD61-PE抗体各30 min,PBS洗涤2遍后重悬,流式细胞仪检测Akt表达水平。P-Akt指数(%) = MFIADP/(MFIADP+MFIresting)×100 (%)。

结 果

入组对象的一般临床资料特征 在入组的208例冠心病患者中,85例(40.9%)患者的CYP2C19基因型为CYP2C19*1/*1,93例(44.7%)患者的CYP2C19基因型为CYP2C19*1/*2和CYP2C19*1/*3,30例(14.4%)患者的CYP2C19基因型为CYP2C19*2/*2和CYP2C19*2/*3。根据CYP2C19基因型,将入组对象分为3组:0LOF(CYP2C19*1/*1),1LOF(CYP2C19*1/*2和CYP2C19*1/*3),2LOF(CYP2C19*2/*2和CYP2C19*2/*3)。未检测到CYP2C19*3/*3及无2个以上LOF。表1显示3组对象中性别、年龄、体质指数(bodymassindex,BMI)、药物治疗差异均无统计学意义。并且高血压、高血脂、糖尿病、陈旧性心梗的患病率及抽烟数差异均无统计学意义。生化指标中ALT、AST、ALP、GGT及eGFR(肾小球滤过率)也无明显差异,血小板计数无差异。

血小板反应性随携带CYP2C19LOF的数目增加而升高 图2显示,2个CYP2C19的携带者血小板聚集率与不携带者相比显著增高(75.1%±13.7% vs.39.9%±19.3%,P<0.001),与携带1个LOF者相比差异也有显著统计学意义(75.1%±13.7% vs. 56.9%±19.7%,P<0.001)。而另外两个组相比后发现1个LOF携带者的血小板聚集率也比不携带者更高(56.9%±19.7%vs.39.9%±19.3%,P<0.001),这些数据显示氯吡格雷负荷量24h治疗后的血小板聚集率因CYP2C19LOF携带数目的增多而增高。

表1 入组对象的临床资料和生化指标

ACEI:Angiotensin-converting-enzyme inhibitors;ARB:Angiotensin Ⅱ receptor blockers;ALT:Alanine aminotransferaseas;AST:Aspartate transaminase;ALP:Alkaline phosphatase;BMI:Body mass index;GGT:Glutamyltransferase;LOF:Loss-of-function;PPI:Proton pump inhibitors;Previous MI:Previous myocardial infarction;eGFR:Estimated glomerular filtration rate; PLT:Platelet count; CCB:Ca chanel blocker.

图2 血小板反应性随SCAD患者携带

VASP-PRI数值随携带CYP2C19 LOF的数目增加而升高 图3显示,2个LOF的携带者VASP-PRI数值明显高于其他两组,与1个LOF携带者相比为(75.8%±9.4%vs.56.6%±18.6%,P<0.001),与不携带者相比为(75.8%±9.4%vs.41.6%±20.6%,P<0.001),而后两组相比为(56.6%±18.6%vs.41.6%±20.6%,P<0.001),数据显示VASP-PRI与血栓弹力图测定的血小板聚集率有着相似的趋势,随CYP2C19 LOF携带数目的增加而升高。

图3 VASP-PRI随SCAD患者携带

P-Akt指数仅在2个CYP2C19 LOF携带者中显著增高 图4显示,携带2个CYP2C19 LOF的患者组P-Akt指数明显高于携带1个LOF组(59.8%±11.7%vs.49.3%±10.3%,P<0.001),且明显高于不携带LOF组(59.8%±11.7%vs.47.5%±11.7%,P<0.001),但是1个LOF组与0个LOF组无明显差异(49.3%±10.3%vs.47.5%±11.7%,P=0.701),数据显示2个CYP2C19 LOF携带者的P-Akt指数显著高于其他两组。

VASP-PRI与P-Akt指数及血小板聚集率的相关性 为了探究氯吡格雷治疗后Gi蛋白亚基αi 和βγ下游信号分子的表达是否具有相关性,经Pearson相关分析发现VASP-PRI与P-Akt指数无相关性(r=0.126,P=0.075,图5A),而VASP-PRI与血小板聚集率显著相关(r=0.672,95%CI:0.590～0.741,P<0.001,图5B)。

NS:No significance.

图4 氯吡格雷治疗后P-Akt指数仅在携带2个CYP2C19

LOF的SCAD患者中显著增高

Fig 4 Postclopidogrel P-Akt index was only elevated in patients with SCAD carrying twoCYP2C19 LOF carrying

A:Relationship between VASP-PRI and P-Akt index;B:Relationship between VASP-PRI and postclopidogrel platelet aggregation.

图5 氯吡格雷治疗后VASP-PRI与P-Akt指数及血小板聚集率的相关关系

Fig 5 Relationships between VASP-PRI and P-Akt index,and between VASP-PRI and platelet aggregation after the treatment of clopidogrel

讨 论

本研究第一次探讨中国稳定型心绞痛患者CYP2C19基因多态性对于氯吡格雷治疗后血小板P2Y12-Gi信号通路分子机制的影响。我们首先从分子水平选择最具代表性的2个下游信号蛋白VASP和Akt共同验证CYP2C19基因多态性对于血小板反应性的影响,其中VASP-PRI随着患者携带CYP2C19 LOF数目的增多而增高,而P-Akt的表达仅在携带2个CYP2C19 LOF的患者中显著增高。虽然VASP-PRI与P-Akt指数无线性相关关系,但是在携带2个LOF的患者血小板内的其表达水平差异均有统计学意义,进一步证实了CYP2C19基因多态性影响氯吡格雷疗效的作用途径。

CYP2C19基因型在不同的人群中有着明显的种族差异,携带CYP2C19 LOF的人数占总人数的35%～45%,在非洲和欧美人中占25%～35%,在东亚人中占55%～70%[11]。5%的非洲及欧美人携带2个LOF(氯吡格雷慢代谢型),而在东亚人的慢代谢型患者中该比例为10%～20%[12]。东亚人群CYP2C19等位基因缺失的频率显著高于西方人群,但是东亚冠心病患者接受氯吡格雷治疗后的心血管不良事件发生率并不高于西方患者[11-13]。我们之前的研究发现中国60.6%的冠心病患者拥有1个或2个CYP2C19 LOF,虽然血小板反应性随LOF的数目增加而升高,但只有携带2个LOF的患者临床预后较差[7-8],还需要更深入研究氯吡格雷治疗后血小板的活性状态及其内部反应,以此更好地说明CYP2C19对于氯吡格雷疗效的影响。本研究以氯吡格雷特异性抑制血小板P2Y12受体进而降低血小板活性的信号通路,证明携带LOF的患者血小板活性并未得到抑制的分子机制,并进一步解释了这个临床现象的内部原因。

动脉粥样硬化斑块破裂后血小板的活化在急性冠脉综合征的发生发展过程中起到了至关重要的作用,因此抗血小板活化是冠心病治疗的基础[14]。血小板受体P2Y12在ADP被激活后,其G蛋白αi亚基介导的VASP去磷酸化使得糖蛋白GPⅡb/Ⅲa处于活化构型,易与纤维蛋白原结合促进了血小板聚集与黏附[15-17]。然而,其另一个亚基βγ介导的Akt磷酸化在血小板的多个活化过程(聚集、分泌、黏附、铺展及回缩)中发挥着非常重要的作用[18-21]。VASP因其涉及的过程较少而敏感性更高,由数据可以看出VASP-PRI随着CYP2C19 LOF数目的增多而增高;而Akt几乎涉及血小板活化的所有过程,并发挥重要作用,敏感性可能相对较弱,P-Akt仅在携带2个CYP2C19 LOF的患者中升高,因Akt磷酸化诱导的血小板激活在ACS发展及预后中不可或缺的作用解释了这部分患者不良心血管事件发生率较高的原因。

本研究存在样本量不大,未在所选人群中进行长期预后随访,选择的中国人群携带LOF频率远远高于西方人群,因此本组研究结果并不能确证CYP2C19基因多态性与血小板内反应状态的必然关系,这些有待于进一步设计大规模、随机、前瞻性的临床研究来验证。

本研究首次以特异性的血小板信号通路证明CYP2C19基因型对于氯吡格雷疗效的影响,分析了VASP及Akt在携带不同数目CYP2C19 LOF患者中的变化趋势,阐明了此通路对冠心病的病理生理及预后的潜在作用,为日后研究抗血小板治疗提供了新的线索。

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Effect ofCYP2C19 polymorphism on platelet P2Y12receptor signaling pathway in Chinese patients with stable coronary artery disease after the treatment of clopidogrel

XU Hua-Jie, ZHAO Xin, WU Hong-yi, FAN Bing△

(DepartmentofCardiology,ZhongshanHospital,FudanUniversity,Shanghai200032)

Objective To investigate the molecular mechanism of the effect ofCYP2C19 polymorphism on the efficacy of clopidogrel in patients with stable coronary artery disease (SCAD).Methods A total of 208 patients with coronary artery disease undergoing percutaneous coronary intervention (PCI) were included.TheCYP2C19 variant alleles were detected by gene sequencing.Platelet reactivity was assessed by thrombelastograph at 24 h after 300 mg clopidogrel loading.P2Y12-Gi signaling was measured by flow cytometric analysis of vasodilator-stimulated phosphorylation-platelet reactivity index (VASP-PRI) and Akt phosphorylation (P-Akt) index. Results Among 208 patients,40.9% were classified asCYP2C19*1/*1 (non-carriers) with no loss-of-function (LOF),44.7% asCYP2C19*1/*2 orCYP2C19*1/*3 (one LOF carriers) and 14.4% asCYP2C19*2/*2 orCYP2C19*2/*3 (two LOF carriers).Both postclopidogrel platelet aggregation and VASP-PRI increased by the presence of one LOF allele,and were even more pronounced with the presence of two LOF alleles.In contrast,P-Akt index only increased in two LOF carriers.VASP-PRI was positively associated with postclopidogrel platelet aggregation (r=0.661,P<0.001),but not with P-Akt index.Conclusions The twoCYP2C19 LOF carriage was associated with more active state of P2Y12-Gi signaling in postclopidogrel platelet in Chinese patients with SCAD.

stable coronary artery disease;CYP2C19; clopidogrel; P2Y12; VASP; Akt;platelet reactivity

国家自然科学基金(81300152)

R541.4

A

10.3969/j.issn.1672-8467.2017.04.003

2016-12-14;编辑:段佳)

△Corresponding author E-mail:fan.bing@zs-hospital.sh.cn

*This work was supported by the National Natural Science Foundation of China (81300152).