【作者】 张弛；

【导师】 杨向军；

【作者基本信息】 苏州大学 ， 内科学（心血管病学）， 2019， 博士

【摘要】 目的:心房颤动(房颤)是世界上导致卒中和心力衰竭的主要原因之一。CHA2DS2-VASc评分≥1分的男性以及≥2分的女性患者需要抗凝治疗。目前抗凝药物层出不穷,针对的靶点也不尽相同,临床上使用最广泛的口服抗凝药物有华法林(warfarin)、达比加群酯(dabigatran etexilate)、利伐沙班等。(1)非维生素K拮抗剂口服抗凝剂(non-vitamin K oral anticoagulants,NOACs)达比加群酯口服后迅速吸收并水解成达比加群(dabigatran),通过与凝血酶(thrombin,Ila)活性位点结合,抑制凝血酶作用于纤维蛋白原。①达比加群血药浓度测定金标准为高效液相色谱-串联质谱法(liquid chromatography-tandem mass spectrometry,LC-MS/MS),但测定方法复杂,本研究首先探索有无简易检测方法替代LC-MS/MS。②根据测得达比加群血药浓度,分析与非瓣膜性房颤患者凝血状态的影响(通过凝血常规及TG反映)。③非瓣膜性房颤患者服用达比加群酯后血药浓度个体差异大,最佳治疗浓度范围尚不明确,研究表明低浓度达比加群导致凝血酶生成(thrombin generation,TG)增多,但高浓度达比加群对患者凝血状态影响尚不明确,本试验予以新西兰兔不同剂量的达比加群酯,模拟人体高浓度达比加群对凝血状态的研究,有助于明确达比加群的最佳治疗浓度,精确指导患者调整达比加群酯的剂量,确保临床用药安全有效。(2)传统的口服抗凝药物华法林上市超过半个多世纪,在临床实践中广泛应用,在瓣膜性房颤中发挥不可替代的作用。达比加群是直接凝血酶抑制剂,能迅速抑制凝血瀑布,但在瓣膜性房颤患者中对比华法林会增加卒中风险,那么了解达比加群和华法林抗凝效果的差异具有重要意义。因此本论文第二部分收集临床非瓣膜性房颤患者,测定凝血常规及TG,并根据患者年龄、肌酐清除率及CHADS2评分进行亚组分析,结合前瞻性临床试验,为不同年龄、肾功能及CHADS2评分房颤患者临床用药提供更多的实验证据。(3)蛋白C(protein C,PC)和蛋白S(protein S,PS)是初始使用华法林导致继发高凝状态的重要因素,初始使用华法林抗凝时,由于PC和PS的半衰期短,发挥抗凝作用的PC和PS首先被抑制,而半衰期相对较长的II、VII、IX和X因子未受到抑制,导致继发的高凝状态,并且初始华法林剂量越大,血液高凝状态越明显。与华法林抗凝机制不同,达比加群起效迅速,前期研究表明低浓度的达比加群即可抑制PC的活化,继发导致对Ⅴa和Ⅷa因子的降解减弱,最终低浓度达比加群矛盾性促进TG。PS与PC发挥作用类似,联系紧密,达比加群对PS的影响尚不明确。本研究第三部分将研究达比加群对PS是否具有直接作用,其次在PS缺乏条件下研究达比加群对TG的影响,通过正反两方面进行验证,完善达比加群的基础研究。方法:(1)第一部分实验将18只普通级新西兰兔随机分成三组,分别灌胃5mg/kg,10mg/kg达比加群酯10mL及等体积溶剂。于服药前,及服药后0.5、1、1.5、2、3、4、6、8、12、24h采集血浆样本。收集46例服用达比加群酯110mg达稳态的非瓣膜性房颤患者,于下一次服药前采集血浆样本,样本以3000rpm离心10min后获得乏血小板血浆冻存于-80℃超低温冰箱。将新西兰兔及患者血浆样品37℃水浴解冻后,通过LC-MS/MS及蛇静脉酶发色底物法(ecarin chromogenic assay,ECA)分别检测达比加群血药浓度,做相关性及偏倚性分析。同时检测新西兰兔及非瓣膜性房颤患者凝血常规和自动校准凝血酶生成实验(calibrated automated thrombogram,CAT),分析低浓度达比加群对非瓣膜性房颤患者凝血状的影响。最后应用新西兰兔模拟高浓度达比加群对患者凝血状态的影响,根据不同浓度的达比加群对上述指标的影响明确达比加群的最佳治疗浓度。(2)第二部分实验收集104例非瓣膜性房颤患者,其中46例服用达比加群酯110mg达稳态,36例服用华法林,国际标准化比率(international normalized ratio,INR)控制在2-3之间,22例患者暂未服用抗凝药物作组间对照。汇总患者的基线资料进行匹配。于下一次服药前抽血,置于枸橼酸钠抗凝管中,3000rpm离心10min后获得乏血小板血浆,分装后冻存于-80℃超低温冰箱。将患者血浆样品37℃水浴解冻后,分别检测凝血常规、CAT、凝血酶原片段(prothrombin fragment 1+2,F1+2)及凝血酶-抗凝血酶复合物(thrombin-antithrombin complex,TAT),并根据患者年龄、肌酐清除率及CHADS2评分进行亚组分析,将结果结合前瞻性临床研究进行对比,分析真实世界中达比加群对比华法林在不同年龄、肾功能及CHADS2评分房颤患者中对凝血常规和TG影响。(3)第三部分实验将正常离体血浆、PS缺乏血浆及PC缺乏血浆中加入不同浓度的达比加群,检测CAT及F1+2,分析TG在正常离体血浆、PS缺乏血浆及PC缺乏血浆中的差异。接着通过凝固法检测正常离体血浆和PS缺乏血浆中Ⅴ、Ⅷ、PC和PS活性,随后通过酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)分析达比加群对PS和活化蛋白C(activated protein C,APC)的影响,对两种方法测定的PS结果进行对比分析。最后将上述结果通过蛋白质免疫印迹(westernblot,WB)进行验证,检测正常离体血浆、加入达比加群的离体血浆及PS缺乏血浆中Va因子降解片段(FVa307-506)和未被降解Va因子水平(FVaHC),明确达比加群对PS的作用。结果:(1)采用LC-MS/MS及ECA检测新西兰兔及非瓣膜性房颤患者达比加群血药浓度,显示两种方法所测结果具有高度相关性。Bland-Altman一致性分析提示在非瓣膜性房颤患者中,ECA能较好预测血浆达比加群浓度,可用于临床实践,而在新西兰兔血浆中,ECA低估达比加群血药浓度。新西兰兔凝血常规示,活化部分凝血活酶时间(activated partial thromboplastin time,APTT)与达比加群浓度低度相关(r2=0.3468),凝血酶时间(thrombintime,TT)与达比加群浓度中度相关(r2=0.7179),凝血酶原时间(prothrombintime,PT)因低于检测下限,未能行相关性分析。患者血浆中,APTT、TT及PT与达比加群浓度有相关性(r2=0.5794,r2=0.7623,r2=0.6384),其中TT与低浓度达比加群相关性较高。CAT提示随着达比加群血药浓度的升高,血液凝固延迟时间(Lagtime)相应延长,然而在人血浆中代表TG的内源性凝血酶潜能(endogenous thrombin potential,ETP)及凝血酶生成峰值(maximum thrombin generation,Cmax)同时增多。在新西兰兔血浆中,ETP及Cmax先随达比加群血药浓度升高而升高,在150ng/mL出现峰值后逐渐降低。(2)收集的104例非瓣膜性房颤患者基线资料匹配。服用华法林的非瓣膜性房颤患者PT(18.5-34.9s)及INR(1.64-3.23)较服用达比加群酯组(PT:10.8-19.8s,INR:0.94-1.7)明显升高。达比加群组TT(15.9-215s)延长更明显,只有1例患者TT值在正常值范围内,其余均超过正常参考值上限,并且组内差异大。APTT在两组中均有延长(达比加群酯组:27.9-86.5s,华法林组:16.2-75.4s),APTT、纤维蛋白原(fibrinogen)及抗凝血酶Ⅲ(antithrombin Ⅲ,ATⅢ)两组间无统计学差异(P>0.05)。TG实验示Lagtime:对照组2.42±0.23min,达比加群酯组:4.44±1.02min,华法林组:4.65±1.5min,华法林及达比加群均延长Lagtime,两组间无统计学差异(P>0.05)。但华法林较达比加群进一步抑制ETP、Cmax、F1+2和TAT。根据患者的年龄、肌酐清除率及CHADS2行亚组分析,各组中华法林较达比加群对TG抑制更显著。(3)正常离体血浆分别加入0、0.5、2、12、35、70、141、283、566ng/mL的达比加群后,CAT结果示随着达比加群浓度的升高,Cmax(TG生成峰值)和ETP(TG总量)矛盾性的升高,在70ng/mL达比加群条件下达到最高峰,该现象在PS或PC缺乏血浆中未能见到,同时通过F1+2验证了CAT实验的结果。凝固法检测正常离体血浆及PS缺乏血浆随着达比加群浓度升高,Ⅴ和Ⅷ活性降低,而PC和PS活性升高,但ELISA结果表明随着达比加群浓度升高,PS及APC水平下降,继发导致WB结果中Va因子的裂解减少。结论:(1)ECA检测达比加群血药浓度与LC-MS/MS检测结果高度相关且操作简易,在临床实践中可作为LC-MS/MS的替代实验。凝血常规能够在无法快速获取达比加群血药浓度情况下起到重要提示作用。非瓣膜性房颤患者低浓度达比加群矛盾性促进TG,新西兰兔TG出现峰值后逐渐降低,患者达比加群最佳治疗窗是否在拐点之后,需要在临床上进一步论证。(2)本实验首次研究达比加群对比华法林对非瓣膜性房颤患者凝血常规和TG的影响。华法林同时抑制内源性和外源性凝血途径,达比加群明显抑制凝血酶,两组药物对内源性凝血途径抑制效果相似。华法林对TG抑制显著强于达比加群。此外,根据患者年龄、肾功能及CHADS2评分行亚组分析,在各组中,达比加群酯可选用统一剂量,达到预防缺血性卒中不劣于华法林的效果。(3)低浓度的达比加群抑制PS,导致Ⅴa,Ⅷa因子降解减少,矛盾性促进TG生成。达比加群作为特异性凝血酶抑制剂,如何作用于PS有待进一步研究,PS缺乏患者应用达比加群酯时是否与正常患者有所区别需要进一步论证。更多还原

【Abstract】 Objective:Atrial fibrillation(AF)is the leading cause of stroke and heart failure in theworldwide.AF patients who are at risk of stroke(CHA2DS2-VASc score≥1 in men or CHA2DS2-VASc score≥2 in women)would benefit from anticoagulation.Nowadays,plenty of anticoagulants are widely used in clinical practice,such as warfarin,dabigatran etexilate,rivaroxaban and so on.1.Dabigatran etexilate could be absorbed rapidly and hydrolyzed to dabigatran after administration.Dabigatran,as a direct thrombin inhibitor,could suppress the transformation of fibrinogen to fibrin by reversely binding to the active site of thrombin.Firstly,liquid chromatography-tandem mass spectrometry(LC-MS/MS)is the gold standard for detecting plasma dabigatran concentrations and shows high sensitivity and accuracy.However,the method is complicated.It is important to find a simple method which could replace LC-MS/MS.Secondly,we would demonstrate the effect dabigatran on thrombin generation(TG)and coagulation assays in patients with non-valvular atrial fibrillation(NVAF).Finally,NVAF patients treated with dabigatran etexilate have a wide range of concentrations.The therapeutic concentrations of dabigatran remain unclear.Previous studies demonstrated that low concentrations of dabigatran could increase TG.It is unknown that how the high concentrations of dabigatran affect the TG and coagulation assays.Rabbit were treated with different doses of dabigatran etexilate.It would be helpful to know the therapeutic concentrations of dabigatran in human beings.The results could guide patients to use dabigatran etexilate properly.2.Warfarin has been applied in clinical practice for more than half a century.Owing to the characteristics of anticoagulation,warfarin plays an important role in patient with valvular atrial fibrillation.New oral anticoagulants(NOACs)were introduced recently which could inhibit coagulation factor specificity.However,NOACs are not indicated for patients with valvular atrial fibrillation.It is essential to know the difference between NOACs and warfarin.In this part,we measured the coagulation assays combined with TG in patients with NVAF.In addition,we compared coagulation status between dabigatran group and warfarin group according to the age,renal function,and CHADS2 score.The results would provide more evidence for drug selection in clinical practice.3.Warfarin would induce a transient hypercoagulability by inhibition of vitamin-K-dependent coagulation factors,protein C(PC)and protein S(PS).The half-life of Ⅱ(72h),Ⅶ(6-8h),Ⅸ(48h)and Ⅹ(36h)factors,PC(6-7h)and PS(42h)is different.PC has a shorter half-life than other factors,so PC levels fall quickly in response to warfarin therapy.An even greater temporary hypercoagulability occurs when a large initial dose of warfarin is administrated.The anticoagulant effect of dabigatran is different from warfarin.Previous studies showed that low concentrations of dabigatran paradoxically increased TG.Dabigatran inhibits PC more aggressively than thrombin at low concentrations resulting in the decrease of FVa degradation.The relationship between PS and PC is close.We are wondering if PS participates in the paradoxical enhancement of TG after administration of dabigatran etexilate.In this part,we would study the mechanism of PS in the paradoxical enhancement of TG.The results could improve the basic research of dabigatran.Methods:Firstly,18 rabbits were randomly divided into three groups:group 1 in which rabbits were treated with 5mg/kg dabigatran etexilate(n=6);group 2 in which rabbits received 10mg/kg dabigatran etexilate(n=6);control group in which rabbits received equivalent volume of solution(n=6).Blood samples were drawn from the ear auricular artery using a 23-gauge needle and collected pre-dose,0.5,1,1.5,2,4,6,8,12,and 24h post oral administration of a single dose of dabigatran etexilate.Platelet-poor plasma was obtained by centrifugation at 3,000g for 10min and stored at-80℃ until analysis.Blood samples collected from 46 patients treated with dabigatran etexilate 110mg twice daily for more than three days before the next dose.Platelet-poor plasma was obtained by centrifugation at 3,000g for 10min and stored at-80℃ until analysis.Rabbit or human plasma samples were thawed at 37℃ for 5min.The concentrations of dabigatran were detected by LC-MS/MS and ecarin chromogenic assay(ECA).The relationship and difference were analyzed.Then,calibrated automated thrombogram(CAT)was used to determine TG.Based on the results from patients and rabbits,we might definite the therapeutic concentrations of dabigatran.Secondly,a total of 104 patients diagnosed as NVAF were recruited from January 2016 to November 2017 in the department of the First Affiliated Hospital of Soochow University.Blood samples collected from patients treated with adjusted-dose warfarin(INR=2-3)or dabigatran etexilate 110mg twice daily for more than three days before the next dose.Blood samples collected from NVAF patients were used as control.Blood samples were centrifuged at 3,000g for 10min and stored at-80℃ until analysis.Human plasma samples were thawed at 37℃ for 5min.The characteristics of the NVAF patients were collected from the medical records.Coagulation assays combined with CAT,prothrombin fragment 1+2(F1+2)and thrombin-antithrombin complex(TAT)were measured to determine the coagulation status in NVAF patients.At last,we compared coagulation status between dabigatran group and warfarin group according to the age,renal function,and CHADS2 score.Thirdly,normal,PS-,and PC-deficiency plasma were spiked with different concentrations of dabigatran.CAT assay was used to evaluate the effect of PS and PC in the process of TG.Ⅴ,Ⅷ factors,PC and PS activities were detected by coagulation method.Free PS and activated protein C(APC)levels were measured by enzyme linked immunosorbent assay(ELISA).TG was induced.Several reaction time points were set up in PS-deficiency plasma and normal plasma spiked with or without 70ng/mL dabigatran according to CAT results.The reaction was stopped with 13μL of 0.5M sodium citrate buffer(pH4.3).FVaHC and its degradation product FVa307-506 were detected by western blot(WB).Results:Firstly,correlation between LC-MS/MS and ECA was performed,and excellent correlations were shown in both rabbit plasma(r2=0.9097)and human plasma(r2=0.9372).ECA could function as a substitute for LC-MS/MS in clinical practice,whereas ECA would underestimate plasma concentrations of dabigatran in rabbits.For rabbits,activated partial thromboplastin time(APTT)and thrombin time(TT)ratio showed low and medium correlations with plasma dabigatran concentrations(r2=0.3468,r2=0.7179 respectively).The association between dabigatran concentrations and prothrombin time(PT)assay could not be assessed,because PT values were low.In human plasma,APTT,TT and PT showed correlation with plasma dabigatran concentrations(r2=0.5794,r2=0.7623,r2=0.6384 respectively).The results from CAT assay showed that Lagtime increased in line with the concentrations of dabigatran.However,TG was paradoxically enhanced both in rabbit and human plasma at low concentrations of dabigatran.TG was suppressed when the plasma dabigatran concentrations exceeded 150ng/mL of dabigatran in rabbits.Secondly,the characteristics of 104 NVAF patients were collected and well matched.PT(18.5-34.9s)and INR(1.64-3.23)were prolonged in the warfarin group than in the dabigatran group(PT:10.8-19.8s,INR:0.94-1.7).While,TT increased obviously in the dabigatran group(15.9-215s).Among patients treated with dabigatran etexilate,only one patient had normal TT.There were significant differences within groups.Although APTT was prolonged in dabigatran group(27.9-86.5s)and warfarin group(16.2-75.4s),there was not significantly different.In addition,there was no statistical difference in fibrinogen and antithrombin Ⅲ(AT Ⅲ).The prolong of Lagtime were similar in warfarin group(4.65±1.5min)and dabigatran group(4.44±1.02min).The endogenous thrombin potential(ETP),maximum TG,F1+2,and TAT were significantly higher in the dabigatran group than in the patients treated with warfarin.What is more,these TG markers were still significantly higher in the dabigatran group regardless of the age,renal function and CHAD S2 score.Thirdly,normal plasma was spiked with different concentrations of dabigatran(0、0.5、2、12、35、70、141、283、566ng/mL).There was a gradual dose-dependent increase of TG in normal plasma,whereas,TG was suppressed at higher concentrations of dabigatran.However,the paradoxical enhancement of TG was absent in PS-and PC-deficiency plasma.F1+2 confirmed the results from CAT assay.The activities of Ⅴ and Ⅷ factors decreased with the concentrations of dabigatran,but the activities of PC and PS increased.However,the results detected by ELISA assay were quite different.Low concentrations of dabigatran could inhibit PS levels and PC activation.Its degradation product FVa307-506 decreased in plasma treated with low concentrations of dabigatranConclusions:Firstly,LC-MS/MS is the gold standard for detecting plasma dabigatran concentrations whether in rabbit and human plasma and shows high sensitivity and accuracy.ECA precisely predicted the concentrations of dabigatran and correlated well with LC-MS/MS.EC A could function as a substitute for LC-MS/MS in clinical practice.Coagulation assay plays important role when concentrations of dabigatran are not reached.Low concentrations of dabigatran paradoxically increased TG.TG was suppressed when the plasma dabigatran concentrations exceeded 150 ng/mL of dabigatran in rabbits.Above all,the adjustment of dabigatran remains to be further demonstrated.Secondly,we compared the coagulation status in NVAF patients treated with dabigatran etexilate or warfarin.Warfarin could inhibit both extrinsic coagulation pathway and intrinsic coagulation pathway.Dabigatran inhibit thrombin specifically.The inhibition of TG was stronger in the warfarin group.TG markers were still significantly higher in the dabigatran group regardless of the age,renal function and CHADS2 score.Thirdly,low concentrations of dabigatran paradoxically increased TG because low concentrations of dabigatran could inhibit PS levels resulting the enhancement of Va andⅧa factors.The effect of dabigatran on PS remains unclear,it needs further investigation.更多还原