【作者】 魏玉杰；

【导师】 刘惠亮；

【作者基本信息】 中国人民解放军军医进修学院 ， 心血管内科， 2012， 博士

【摘要】 吸烟是心血管疾病的独立危险因素，可导致内皮功能紊乱，炎症反应增加，血管内血栓形成，易诱发急性心血管事件的发生。其中针对吸烟导致血栓形成机制的研究虽有报道，但是目前尚未阐明。血栓调节蛋白（Thrombomodulin, TM）是内皮细胞表面的一种糖蛋白，作为一个重要的抗凝辅助因子，它与凝血酶以1：1结合，通过激活蛋白C起到抗凝作用。而TM与凝血酶的结合不仅取决于TM量的表达，而且也取决于其与凝血酶在单分子水平的亲和力。目前针对吸烟对TM表达量的研究虽有少量报道，但是研究结果显示吸烟对可溶性TM表达的影响不一致；另外，对于TM与凝血酶在单分子水平的相互作用的研究未见报道。吸烟是否通过影响TM量的表达或者影响其与凝血酶的结合力这条途径导致血管内血栓形成不清楚。研究表明，支架术后患者持续吸烟会增加支架内再狭窄率及心血管事件的发生，然而戒烟可以使冠心病患者的死亡率下降36%。由于吸烟是一种成瘾性疾病，戒除后病人会出现焦虑、烦躁等戒断症状，同时机体里的尼古丁水平下降、从而导致多巴胺水平下降，可能诱发心血管事件发生，而其仅凭个人毅力戒烟成功率很低，故需借助戒烟药物缓解戒断症状，提高戒断率，从而减少心血管事件的发生。伐尼克兰是一种尼古丁乙酰胆碱受体部分激动剂，目前已被全球70多个国家批准为戒烟辅助药。支架术后病人常规服用氯吡格雷抗血小板治疗，防止支架内血栓形成。故有可能支架术后的病人同时服用戒烟药物和氯吡格雷。但是两者间是否有药物相互作用，目前没有循证医学证据。第一部分香烟提取物对血管内皮细胞表面血栓调节蛋白表达的影响目的：研究不同条件下的香烟提取物（Cigarette Smoke Extract, CSE）对人脐静脉内皮细胞（Human Umbilical Vein Endothelial Cell, HUVEC）表面TM量的表达，从而探讨吸烟导致血管内血栓形成的可能机制。方法：参照Nakamura方法制备CSE在30min内用于实验。以0.1%I型胶原酶37℃消化15-20min人脐静脉内皮，分离获得的原代HUVEC用低血清内皮细胞专用培养基培养，37℃、5%CO2孵箱内培养至融合后传代；用VIII因子相关抗原鉴定HUVEC，将第2-3代细胞用于实验。将第3代HUVEC接种至6孔板，用不同浓度CSE（0、0.5%、1%、2.5%、5%）同一时间（6小时）及同一浓度5%CSE不同时间（0h、6h、10h、12h、24h）孵育、收集细胞，分别采用流式细胞仪计数和实时荧光定量PCR（Real-time PCR）方法检测TM的蛋白量和mRNA水平的表达。结果：（1）与对照组相比，6小时不同浓度（0.5%、1%、2.5%、5%）CSE降低了HUVEC表面TM在mRNA和蛋白水平的表达（P＜0.01）；mRNA水平组间比较除了0.5%CSE和1%CSE组没有统计学差异，其它两两浓度比较均有显著差异（P＜0.001）；蛋白水平组间比较，5%CSE组与0.5%CSE组有统计学差异（P＜0.05）,其它浓度组间比较无统计学差异。（2）与对照组比较，同一浓度(5%) CSE不同时间（6h、10h、12h、24h、）明显降低了HUVEC表面TM在mRNA和蛋白水平的表达（P＜0.01)；并且在12小时以内TM的表达随着时间延长逐渐降低，至24小时以后TM表达不再继续下降。结论：CSE可下调HUVEC表面TM的表达，并呈量-效关系。12小时以内呈时-效关系。第二部分香烟提取物对血栓调节蛋白与凝血酶结合力的影响目的：利用单分子力谱法检测CSE处理后的TM与凝血酶单分子水平结合力，从而探讨吸烟导致血管内血栓形成的可能机制。方法：1.从HUVEC中提取总RNA，反转录为cDNA，构建TM-GFP质粒并转染至COS-7细胞分组测力：（1）空白质粒对照组（GFP-thr group）（2）TM-GFP对照组（TM-thr group）（3）5%CSE孵育空白质粒细胞组（CSE-GFP-thr group）（4）5%CSE孵育TM-GFP细胞1小时组（CSE-TM-thr1h group）（5）5%CSE孵育TM-GFP细胞6小时组（CSE-TM-thr6h group）每组测力后均加入鼠抗人特异单克隆抗TM抗体行封闭阻断实验。在2.0±0.5×103pN/s的加载速率范围下，对荧光显微镜下观察各组细胞所获得的TM荧光融合蛋白聚集区，用凝血酶修饰的原子力显微镜(atomic forcemicroscope, AFM)探针反复在细胞表面下压-回拉，随机获得力-距离曲线，统计得出针尖和细胞间亲和力的高斯分布及成键几率。2.用流式细胞仪计数法检测5%CSE孵育TM-GFP转染后的COS-7细胞1h、6h的荧光强度。结果：（1）TM-Thr对照组高斯拟合的结合力：TM与凝血酶最可几的相互作用力的大小为60.90311±0.81723pN，TM与凝血酶的AFM成键几率为22.58±3.95%，抗体阻断后成键几率为2.58±2.0%。（2）与TM-Thr group比较， GFP-Thr group，CSE-TM-Thr1h group，CSE-TM-Thr6h group，CSE-GFP-Thr group四组AFM成键几率明显减低，P<0.0001；但四组间两两比较无统计学差异。（3）用流式细胞仪计数法检测5%CSE孵育TM-GFP转染后的COS-7细胞1h、6h的荧光强度表达结果：和对照组比较，5%CSE组1h、6h荧光强度没有统计学差异。结论：在CSE孵育COS-7细胞1小时、6小时不影响TM荧光量的表达下，CSE可明显减少TM与凝血酶的成键几率。结合第一部分研究提示：吸烟可能通过下调TM量的表达并减少其与凝血酶结合几率，抑制了TM的抗凝作用，从而导致血管内血栓形成。第三部分伐尼克兰对氯吡格雷抗血小板作用影响的研究目的：通过观察戒烟药物伐尼克兰对氯吡格雷抗血小板作用的影响，企图为戒烟药物和抗血小板联合用药的安全性方面提供循证医学证据。方法：连续选取自2010年7月至2012年3月在武警总医院心内科住院的择期行经皮冠状动脉介入治疗（Percutaneous coronary intervention, PCI）术的冠心病吸烟患者136例，签署知情同意书后随机分为两组：常规治疗组和常规治疗加伐尼克兰组。所有入选病人均于术前给予阿司匹林100mg/d,氯吡格雷300mg/d，随后给予75mg/d服用第三天随机给与或不给与伐尼克兰治疗，两组均提供戒烟方法和咨询随访。记录所有病人的冠心病危险因素、合并疾病及合并用药等情况。服用氯吡格雷前、伐尼克兰前抽血化验血生化（血糖、血脂、肝肾功、尿酸、心肌标志物等）、血小板聚集率、血小板反应指数(platelet reactivityindex, PRI)及服用后第7天、14天抽血化验血肌酐、心肌标志物、血小板聚集率、PRI，记录不良事件和严重不良事件。结果：1.两组基线水平比较无差别。2.常规治疗组和伐尼克兰组在服用伐尼克兰2周的血小板聚集率及PRI比较（1）PRI%:服用氯吡格雷前80.08±5.60vs79.94±5.18，P=0.893；服用伐尼克兰前55.21±14.87vs53.74±14.47，P=0.591；服用伐尼克兰1周41.87±17.05vs40.16±15.81，P=0.579；服用伐尼克兰2周43.01±13.59vs42.18±11.27，P=0.723。两组PRI均随着时间的延长明显降低，具有统计学差异（P=0.000）。（2）血小板聚集率：入院时：服用氯吡格雷前44.13±7.89vs43.67±7.31，P=0.744；服用伐尼克兰前26.53±11.27vs24.92±10.75，P=0.433；服用伐尼克兰1周21.42±10.56vs20.45±9.75，P=0.612；服用伐尼克兰2周23.61±10.09vs23.34±10.64，P=0.893。两组血小板聚集率均随着时间的延长明显降低，具有统计学差异（P=0.000）。3.（1）2周两组肾功能比较常规治疗组和伐尼克兰组的肾小球滤过率（estimated glomerular filtrationrate，eGFR）在各时间点均无统计学差异。（2）不良事件和严重不良事件情况伐尼克兰组的不良事件较常规治疗组略有增加，但两组无统计学差异；严重不良事件：两组均发生围手术期心梗5例，并未发生支架内血栓等其它严重不良事件。结论：短期观察伐尼克兰与氯吡格雷合用不影响后者的抗血小板作用，是安全的。总结：基础研究提示：通过下调TM量的表达及影响其与凝血酶结合力，可能是吸烟导致血管内血栓形成的机制之一。临床研究提示：冠心病行PCI术的吸烟患者服用戒烟药伐尼克兰不影响氯吡格雷的抗血小板作用，不需调整用量。更多还原

【Abstract】 Cigarette smoking is independent risk factor of cardiovscular disease. Numerousstudies have shown that smoking cause signigicant endothelial dysfunction,inflammatory state, triggering of coronary thrombosis, coronary arteryspasm.Accordingly, smoking is associated with significantly increased rates of acutecoronary syndrome. However, the mechanism by which smoking cause arterialthrombosis remains unknown. Thrombomodulin (TM) is constitutively expressed onthe endothelial cell surface.The major function of TM is to form a high affinity1:1complex with thrombin.The complex of TM/Thrombin has several important rolesincluding activation of protein C and thrombin activatable fibrinolysis inhibitor,aswell as inhibition of inflammation.Thus,TM has a critical effect on anticoagulationand anti-inflammation.Functional deficiency of TM could enhance thrombosisformation. The combination of TM and thrombin depends not only on the amount ofexpression of TM but also on the single-molecule interactional force between TMand thrombin. Several studies show that the effects of cigarette smoking on TMexpression are inconsistent. Moreover, the single-molecule interactional forcebetween TM and thrombin is not reported. However, it is not clear the way leading tointravascular thrombosis whether cigarette smoking affect the expression of TM oraffect the interaction with thrombin.A research showed that continued smoking after successful percutaneouscoronary intervention (PCI) is associated with an increased risk of restenosis.However, smoking cessation can make a36%reduction in crude relative risk (RR) ofmortality for patients with CHD. Hence current management guidelines nowadvocate smoking cessation, in addition to controlling hypertension and dyslipidemia,as part of an overall cardiovascular risk reduction strategy. Varenicline is a novelselective nicotinic acetylcholine receptor partial agonist that has been approved in over70countries worldwide as an aid to smoking cessation. Clopidogrel is widelyused by patients with coronary artery disease undergoing PCI. The relationshipbetween smoking and cardiovascular disease increases the prospect of patientsreceiving smoking cessation therapy and Clopidogrel concomitantly in clinicalpractice. Plasma protein binding of Varenicline is low(≤20%) and independent of ageor renal function. The major route of clearance for varenicline is renal excretion.Clopidogrel, a prodrug, is metabolized by2consecutive cytochrome P450-dependentsteps to its active metabolite, which binds irreversibly to the platelet P2Y12receptor.The likelihood of a clinically relevant drug-drug interaction between varenicline andClopidogrel was considered to be low; nevertheless, the possibility of an interactionbetween these2drugs is lack of clinical evidences.Part Ⅰ: Effect of cigarette smoke extract on the expression of Thrombomodulinin human endothelial cellsObjective To observe the expression of thrombomodulin in HUVECs underdifferent cigarette smoke extract conditions.Methods CSE was prepared by a modification of Nakamura’s method. HUVECwere isolated by0.1%type I collagenase digestion for15-20min at37℃, andcultured in low serum endothelial cell medium. After passage cultivation, we usedthe factor VIII related antibody to identify endothelial cell. The third generation ofHUVECs were incubated respectively with0,0.5%,1%,2.5%and5%CSE for6hours or exposed to5%CSE for0,6,10,12,24hours to determine the expressionchanges of TM protein and mRNA expression in HUVECs. RT-PCR and Flowcytometric analysis techniques were used for detecting TM mRNA and protein.Results After6-hour exposure to CSE, the protein level of of endothelial TM indifferent concentrations (0.5%,1%,2.5%,5%) reduced significantly (62.06±3.87,56.22±3.93,55.81±3.32,53.70±3.95) compared to control group (70.78±6.55)(P<0.01). and the mRNA level of endothelial TM also decreased significantly(0.1859±0.0139,0.1776±0.0156,0.0853±0.0136,0.0571±0.0123) compared tocontrol group (0.2550±0.0345)(P<0.01). After stimulation with5%CSE for0,6,10, 12,24hours, the levels of TM mRNA and protein decreased over time and reachedthe peak at12hours (0.0348±0.0148,30.51±0.61), which were significantly lowerthan that of control group (0.2241±0.0222,68.98±3.56)(P <0.001).Conclusion CSE significantly decreased the expression of TM in aconcentration-dependent fashion in Human Umbilical Vein Endothelial Cell. Thatsuggests cigarette smoking maybe according this way to lead to intravascularthrombosis.Part Ⅱ Study of the effect of cigarette smoking extract on the interaction betweenThrombomodulin and thrombin by live-cell single-molecule force spectroscopyObjective To study the effect of CSE on the single-molecule interactional forcebetween thrombomodulin and thrombin by live-cell single-molecule forcespectroscopy.Methods1. CSE was prepared by a modification of Nakamura’s method.Total RNA was extracted from HUVECs and construct the plasmid of TM-GFP. COS-7cells were transfected with the recombinant plasmid TM-GFP and the expression of TM-GFP was detected by fluorescence microscopy and laserscanning confocal microscopy. The transfected COS-7cells were grouped (1)GFP-thrombin group (2) TM-thrombin group (3) CSE-GFP-thrombin group(4) CSE-TM-thrombin-1h group (5) CSE-TM-thrombin-6h group. Force measurements with the thrombin modified AFM tips on the living cell surface werecarried out on PicoSPM II with a Pico-Scan3000controller and a larger scanner. The force curves measured in living cells were recorded by PicoScan5software and analyzed by MATLAB R2009aMetlab. Drawn affinity between tip and cell of the Gaussian probability distribution and bonding probility wereanalyzed by Origin7.0software.2. Flow cytometry was used to detect5%CSE on the effects of the expressionTM-GFP on COS-7cells at1hours and6hours.Results1. The single-molecule binding force of Thrombomodulin and thrombin(TM-Thr) was determined60.90311±0.81723pN. The binding probability forTM-Thr was about22.58±3.95%. Antibody blocking binding probability for TM-Thr was2.58±2.0%.2. Compared to TM-THr group, the binding probability for GFP-Thr group，CSE-TM-Thr1h group，CSE-TM-Thr6h group，CSE-GFP-Thr group significantlydecreased (P<0.0001).3. After1-hour and6-hour exposure to5%CSE, there was no significantdifference between5%CSE group and control group in the expression of TM-GFP.Conclusion CSE significantly decreased the binding probability for TM-Thr.Together with the results of PartⅠ, our findings indicate that cigarette smokingdecreased the expression of TM and reduced the binding probability for TM andthrombin to lead to intravascular thrombosis.Part Ⅲ: Influence of varenicline on the antiplatelet action of clopidogrel: therandomized, open-label VACL (Varenicline Clopidogrel) studyObjective This study investigated the effect of varenicline on the antiplateletaction of clopidogrel in patients with coronary artery disease and evaluated the safetyand tolerability of concurrent administration of clopidogrel and varenicline.Methods Eligible smoking patients in hospital, with coronary arterydisease(CAD) undergoing PCI were consecutively included. All patients receivedaspirin (100mg/day) and clopidogrel (loading dose300mg, followed by75mg/day).On3day after received clopidogrel75mg/day, patients will be randomized to receiveeither associated varenicline (varenicline were titrated as follows:0.5mg/d for days1to3,0.5mg twice per day for days4to7, then1mg twice per day) or withoutvarenicline for14days. Clopidogrel effect was tested before associated varenicline,and on7,14day after associated varenicline in both groups by measuring plateletphosphorylated-VASP expressed as a platelet reactivity index (PRI) and plateletaggregation. All patients were documented adverse events and serious adverseevents.Results1.136patients were included. Conventional therapy group was68andvarenicline group was68.11of conventional therapy group failed to return on Day14for blood sampling.7of varenicline group failed to return on Day14for blood sampling.3of varenicline group did not take varenicline accoding to protocol towithdraw. Baseline characteristics were similar in the2groups.2. Changes in PRI and platelet aggregation at baseline, before taking varenicline,on day7, and on day14between the2groups.(1) In the conventional therapy group, PRI was80.08±5.60%,55.21±14.87%,41.87±17.05%and43.01±13.59%respectively at the four time. In vareniclinegroup, PRI was79.94±5.18%,53.74±14.47%,40.16±15.81%,42.18±11.27%respectively at the four time. There was no significant difference between the twogroups.(2) In the conventional therapy group, platelet aggregation was44.13±7.89%,26.53±11.27%,21.42±10.56%and23.61±10.09%respectively at the four time. Invarenicline group, platelet aggregation was43.67±7.31%,24.92±10.75%,20.45±9.75%,23.34±10.64%respectively at the four time. No significant difference wasobserved between the two groups.3. The safety and tolerability of concurrent administration of clopidogrel andvarenicline(1) The status of renal function in2weeks between the2groupsThe data of eGFR were similar in the2groups.(2) The status of adverse events and serious adverse events in2weeks betweenthe2groupsThe adverse events in varenicline group were higher than in conventional therapygroup. However, there was no significant difference between the2groups.The serious adverse events were identical between the2groups.Conclusion Clopidogrel can be safely administrated with varenicline withoutthe need for dose adjustment.更多还原