【作者】 李佳蓓；

【导师】 黄岚；

【作者基本信息】 第三军医大学 ， 内科学， 2013， 博士

【摘要】 背景及目的动脉粥样硬化血栓形成是全球首位的致死、致残病因。抗血小板药物如阿司匹林和氯吡格雷已广泛应用于心血管疾病的一级和二级预防。而抗血小板药物抵抗的出现向现有抗血小板治疗策略提出挑战。依从性不良是抗血小板药物抵抗的常见机制之一。研究表明实验室抗血小板药物抵抗与临床不良事件发生相关。如何治疗抗血小板抵抗是循环研究的热点。当今心血管临床，抗血小板治疗原则同一化用于所有人，忽略个体对抗血小板剂的反应差异。我们刚起步在个体化心血管病治疗的初时代。为降低抗血小板抵抗增加的缺血事件风险，研究者提出以下几种可能策略：1)增加现有抗血小板药物的剂量；2)在现有治疗基础上加用其他的抗血小板药物；3)换用其他的抗血小板药物；4)开发有效的新型抗血小板药物。针对前3个方面在抗血小板药物抵抗中的应用，已有少许研究但结果有争议。血小板活化在急性冠脉综合征(acute coronary syndrome, ACS)的发病中起重要作用。尽管接受合理的治疗，ACS患者再发血栓栓塞事件的风险仍很高，这可能与针对ACS重要发病机制－血小板活化的抗血小板治疗发生抵抗相关。人载脂蛋白A-IV(apolipoprotein, apoA-IV)具有控制食物摄入、调节脂质代谢、抗炎、抗氧化、抗动脉粥样硬化等作用。研究发现，正常血浆凝血块中鉴定出apoA-IV表达；apoA-IV基因多态性与食用富含核桃的低脂肉类后发生血栓的风险相关；常用溶栓药阿替普酶和替奈普酶可以降低apoA-IV水平。综上提示，apoA-IV很可能与血小板活化及血栓形成相关。本研究旨在：1)系统性评价阿司匹林依从性佳的冠心病患者阿司匹林抵抗与主要不良心血管事件风险的相关性，并鉴定个体化治疗在抗血小板药物抵抗患者预后中的有效性；2)探讨临床ACS患者apoA-IV血浆浓度变化；3)利用重组人apoA-IV，从体外水平研究重组人apoA-IV对血小板活化的影响，并探讨其作用的可能机制。旨在发掘治疗ACS血小板活化的新靶点，期望为临床个体化抗血小板药物治疗策略提供新线*本研究受国家自然科学基金资助(No.81100218)索。方法1.电子检索PubMed数据库、EMBASE数据库、Web of Science数据库、Cochrane图书馆，以及手工检索，纳入两组文献：1)前瞻性研究阿司匹林抵抗与不良事件相关性的文献，并确认患者对阿司匹林的依从性；2)随机化研究个体化治疗在抗血小板药物抵抗中的临床效益，主要终点是死亡和支架内血栓形成，并收集所有临床不良事件和出血并发症。对纳入文章进行质量评估后进行定性和定量分析，前者包括临床资料的统计和分析，后者包括异质性分析、meta分析、亚组分析、敏感性分析及发表偏倚。2.纳入115例临床确诊的ACS患者，及68例来自体检中心与病例组年龄和性别匹配的对照个体，收集临床资料，检测并比较两组血小板计数、常规血脂项目(包括总胆固醇、甘油三酯、低密度脂蛋白胆固醇、高密度脂蛋白胆固醇、apoA-I和apoB)、凝血功能(包括凝血酶时间、凝血酶原时间、部分活化凝血活酶时间和纤维蛋白原含量)及血浆apoA-IV的水平。3.利用重组人apoA-IV体外孵育血小板，观察血小板PAC-1表达及纤维蛋白原结合能力的改变。用流式细胞仪检测apoA-IV对血小板α颗粒释放CD62P的影响。利用免疫共沉淀鉴定血小板与apoA-IV的结合靶点；利用GPIIb/IIIa转染的CHO细胞，明确apoA-IV是否通过与GPIIb/IIIa结合而阻断血小板活化；western blotting检测血小板上ABCA1、ABCG1和SRB1的表达。结果1.9项对1889例阿司匹林依从性佳的冠心病患者随访1个月~2.5年，共622例(33.0%)被鉴定为阿司匹林抵抗。且阿司匹林抵抗患者发生不良事件的风险较敏感者显著增高[17.2%vs.9.1%;比值比(95%可信区间)=2.44(1.81–3.30), p <0.00001)]。2.另外7项随机研究中，共纳入12048例PCI术患者，3738例(31.0%)被证实抗血小板抵抗，个体化治疗较常规治疗显著减少抗血小板抵抗患者发生死亡和支架内血栓形成风险[0.5%vs.2.2%;比值比(95%可信区间)=0.25(0.13–0.49), p <0.0001]，总体不良事件也得到改善[5.5%vs.10.0%;比值比(95%可信区间)=0.40(0.20–0.77), p=0.006]，且不增加出血风险(p=0.08)。3. ACS患者血浆apoA-IV水平较对照组显著下降(437.0±157.5μg/mL vs.590.2±183.7μg/mL, p <0.001)。从对照组，到不稳定性心绞痛组(UAP)(457.3±152.9μg/mL),再到急性心肌梗死组(AMI)(311.7±127.8μg/mL)，apoA-IV血浆浓度呈下降趋势。且血浆apoA-IV水平与NYHA心功能分级呈负性相关。NYHA II级和III/IV级患者apoA-IV血浆浓度分别为467.2±142.1μg/mL(vs.对照组, p <0.001)和368.1±170.8μg/mL(vs.对照组, p <0.001)。逐步多变量回归分析表明ACS类型、NYHA分级和血浆纤维蛋白原水平是决定ACS患者血浆apoA-IV水平的重要因素。4.重组人apoA-IV显著抑制人血小板PAC-1表达及纤维蛋白原结合，及抑制活化血小板释放α颗粒内CD62P至血小板表面。免疫共沉淀未在血小板上鉴定出重组apoA-IV的结合靶点。活化GPIIb/IIIa转染的CHO细胞和apoA-IV共孵育后未发现apoA-IV与纤维蛋白原竞争性结合GPIIb/IIIa。Western blotting证实血小板膜表面表达胆固醇转运受体ABCG1和SRB1。结论1.阿司匹林依从性佳的抵抗患者发生主要不良心血管事件(MACEs)的风险较敏感者显著增高。对抗血小板药物抵抗的PCI术患者进行个体化抗血小板治疗，可以显著减少患者随访期间发生临床不良事件的风险，且不增加出血风险。仍需要大规模研究进一步验证个体化抗血小板治疗的有效性，以及发掘有效的新型抗血小板药物。2. ACS患者血浆apoA-IV水平较对照组显著降低，且与病情程度、心功能分级增加、纤维蛋白原含量呈负相关。3.重组人apoA-IV抑制体外人血小板活化。机制可能与apoA-IV作为胆固醇受体，接受血小板膜表面ABCG1和SRB1逆向转运的胆固醇、进而改变血小板膜脂质结构有关。这为临床发掘新型有效的抗血小板剂提供理论依据，及为抗血小板抵抗患者的个体化治疗提供新策略。更多还原

【Abstract】 Background and objectivesAtherothrombosis is the first cause of death and disability globally. Antiplatelet agentssuch as aspirin and clopidogrel have been widely used in primary and secondary preventionof cardiovascular disease. The emergence of antiplatelet resistance challenges the currentantiplatelet guidelines. Poor adherence is a very common reason for antiplatelet-resistance.And it has been confirmed that antiplatelet resistance is associated with increased risk ofadverse events.How to treat antiplatelet resistance is the important concern in cardiovascular research.However, one-size-fits-all still seems to be the current principle regarding antiplatelettreatment in clinical cardiology, and the individual response to antiplatelet agents has beenignored for a long time. We are just entering a new era in personalized cardiovasculartherapies. Many therapeutic strategies have been proposed to decrease the risk of ischemiain patients with antiplatelet resistance. Such strategies that might serve as ways of tailoringantiplatelet therapy include the following:1) increasing the dose of original antiplateletagents,2) adding additional antiplatelet agents, and3) switching to other antiplatelet agentsor other methods of administration (e.g. switching from oral to intravenous administration).Currently, only a few randomized studies about the former three aspects mentioned above,have assessed the efficacy of tailored antiplatelet therapy on clinical adverse outcomes inantiplatelet-resistant patients. However, it remains controversial whetherantiplatelet-resistant patients benefit from a personalized strategy.Platelet activation plays an important role in the pathogenesis of acute coronarysyndrome (ACS). Despite receiving appropriate treatment, the risk of recurrent thromboembolic events in ACS patients remains high; this may be partly explained byantiplatelet resistance.Human apolipoprotein A-IV (apoA-IV) has a variety of physiological roles, whichinclude controlling food intake, regulating lipid metabolisms, anti-inflammation,anti-oxidation, anti-atherosclerosis, and so on. Moreover, in normal plasma clot theexpression of apoA-IV has been identified; there was an association between apoA-IV genepolymorphism and risk of thrombosis in people consuming low-fat meat rich in walnuts;two commonly used thrombolytic agents alteplase and tenecteplase can lower apoA-IVlevels. These evidence could give us a clue that apoA-IV may be related to plateletactivation and thrombus formation.In the study we aimed at:1) systematically evaluating in prospective studies theassociation between aspirin resistance in CHD patients with good compliance to aspirin andrisk of major adverse cardiovascular events, and in randomized studies the efficacy oftailored antiplatelet therapy in antiplatelet-resistant patients on clinical outcomes;2)investigating the apoA-IV plasma concentrations in ACS patients;3) studying the influenceof recombinant human apoA-IV in platelet activation, and exploring the possiblemechanisms. We expected to achieve a novel strategy for antiplatelet therapy.MethodsAn electronic literature search of PubMed, EMBASE, Web of Science and theCochrane Library as well as a hand search of bibliographies was conducted. Two groups ofstudies were included:1) they prospectively investigated the association of aspirinresistance with the risk of adverse cardiovascular events during follow-up in CHD patientswith confirmed compliance;2) they randomized assessed clinical efficacy of personalizedantiplatelet treatment in antiplatelet-resistant patients. The quality of included articles wasevaluated for qualitative and quantitative analysis, the former analysis including statisticsand analysis of clinical data, the latter one including heterogeneity, meta-analysis, subgroupanalysis, sensitivity analysis, and publication bias.In the next part,115ACS patients and68age-and sex-matched control individualswere included. Clinical data and blood samples were collected. Platelet count, routine bloodlipid profile (total cholesterol, triglycerides, low density lipoprotein cholesterol, highdensity lipoprotein cholesterol, apoA-I and apoB), coagulation profile (thrombin time, prothrombin time, activated partial thromboplastin time and fibrinogen) and plasmaapoA-IV levels were measured and compared.In the final part, PAC-1expression and fibrinogen binding in platelets were measuredafter recombinant apoA-IV treatment. CD62P release from α particle was studied by flowcytometry. For detecting the potential mechanism, we identified the acting targets ofapoA-IV on platelets using the co-immunoprecipitation, utilized GPIIb/IIIa transfectedCHO cells to confirm whether apoA-IV binded to GPIIb/IIIa, and detected cholesteroltransporters ABCA1, ABCG1and SRB1expression in platelets by western blotting.ResultsNine prospective studies with a total1,889CHD patients who were followed for1month to2.5years and study sample sizes ranging from86to496patients were identified.Overall,622out of the1,889CHD patients (33.0%) were classified as aspirin resistant withconfirmed aspirin adherence. Aspirin-resistant patients exhibited a significantly higher riskof adverse events compared with aspirin-sensitive patients [17.2%vs.9.1%; OR (95%CI)=2.44(1.81–3.30), p <0.00001].Data were combined across7randomized studies comprising12,048subjects, ofwhom3,738(31.0%) were found to be antiplatelet-resistant. Antiplatelet-resistant patientsprovided with tailored antiplatelet therapy showed less risk of death or stent thrombosisthan those assigned conventional antiplatelet treatment [0.5%vs.2.2%; OR (95%CI)=0.25(0.13–0.49), p <0.0001]. A significant benefit in terms of total adverse event riskreduction was observed during follow-up for tailored vs. conventional antiplatelet therapy[5.5%vs.10.0%; OR (95%CI)=0.40(0.20–0.77), p=0.006]. No statistical difference inbleeding complications was observed between these two groups (p=0.08).Plasma apoA-IV levels in ACS patients were significantly decreased compared to thelevels in control subjects (437.0±157.5μg/mL vs.590.2±183.7μg/mL, p <0.001). Anobvious decreasing trend of plasma apoA-IV levels from the control subjects, to patientswith unstable angina pectoris (UAP)(457.3±152.9μg/mL), to patients with acutemyocardial infarction (AMI)(311.7±127.8μg/mL), was observed. Moreover, plasmaapoA-IV level was negatively associated with New York Heart Association (NYHA)functional class. NYHA class II (467.2±142.1μg/mL, p <0.001) and class III/IV (368.1±170.8μg/mL, p <0.001) patients had statistically decreased levels of plasma apoA-IV when compared to the control subjects. A stepwise multivariate regression analysis identifiedtypes of ACS, NYHA classes, and plasma fibrinogen levels as the most importantdeterminants of plasma apoA-IV levels in ACS patients.Recombinant human apoA-IV significantly inhibited human platelet PAC-1expressionand fibrinogen binding, and decreased the release of CD62P from α particle in activatedplatelets. No targets of recombinant apoA-IV could be identified on platelets byco-immunoprecipitation. Recombinant apoA-IV did not competitively bind to GPIIb/IIIatransfected CHO cells on fibrinogen-coated surface. Significant expression of ABCG1andSRB1was confirmed in platelets by western blotting.ConclusionsIn CHD patients, on confirmed aspirin adherence, aspirin-resistant patients exhibit anincreased risk of MACEs compared with aspirin-sensitive patients. And personalizedantiplatelet treatment for antiplatelet resistance is associated with less occurrence of deathor stent thrombosis and the less risk of total clinical adverse events than conventionaltreatment, without increasing the risk of bleeding complications. Low plasma apoA-IVlevels are decreased in ACS patients, and recombinant human apoA-IV can inhibit humanplatelet activation in vitro. ApoA-IV as a cholesterol acceptor collects cholesterol fromplatelet surface by cholesterol transporters ABCG1and SRB1, thereby affecting plateletmembrane lipid structure, which could be the potential underlying mechanism. This helpsus to explore an effective antiplatelet agent, and a novel strategy for tailored antiplatelettreatment.更多还原