【作者】 张恒；

【导师】 董力；

【作者基本信息】 四川大学 ， 外科学（胸心外科）（专业学位）， 2021， 博士

【摘要】 背景维生素K拮抗剂(VKA)作为机械瓣膜置换术后唯一有效的抗凝药,是目前任何新型抗凝药物无法取代的。华法林作为国内最常用的维生素K拮抗剂,因其安全范围窄,且影响抗凝强度的因素众多,导致抗凝强度容易波动。因为抗凝强度的波动与抗凝治疗的安全性和抗凝相关并发症的发生密切相关,所以,把握不同抗凝治疗强度下的最佳处理方案至关重要。临床上常见的华法林抗凝治疗强度包括下列三种情况:安全范围(最佳抗凝强度),抗凝过度和抗凝不足。目前,国内对于华法林的抗凝治疗并无相应指南,仅有的心内科专家共识也以借鉴西方指南为主,并无国人数据及文献支持。上世纪90年代中期以来,国内已有大量临床研究及数据证实国人抗凝与西方国家的差异,中国人的抗凝最佳强度低于西方人群,其抗凝的主要矛盾是出血,而非血栓栓塞。所以,照搬西方的抗凝指南,应用于中国人群是不合适的。中国人口服华法林的最佳抗凝强度以及抗凝过度的治疗,是目前国内存在争议也十分关注的问题。本研究分三部分,分别探讨适合中国人机械瓣膜置换术后口服华法林抗凝治疗的最佳抗凝强度,抗凝过度的最佳治疗方案,以及抗凝过度的遗传学基础。第一章:心脏机械瓣膜置换术后最佳抗凝强度的临床研究目的:随访我院心脏机械瓣膜置换术后口服华法林抗凝治疗的INR实测值,并计算不同特定抗凝强度范围内不良事件发生率,进而得出机械瓣膜置换术后口服华法林抗凝治疗的最佳抗凝强度。方法:本研究是一项单中心回顾性的研究。根据研究设计的纳入和排除标准,纳入2013年1月至2019年1月我院植入St.Jude机械瓣膜的患者,随访1-6年,随访期间至少每8周进行一次INR监测,并记录终点事件:血栓栓塞和严重出血。通过假设两个连续检测的INR为线性变化关系,从而计算不同INR范围的不良事件发生率来评估中国人双叶机械瓣的最佳抗凝强度。采用相同的方法,对不同瓣膜部位和有无血栓栓塞危险因素进行分组,分别计算不同瓣膜部位有无栓塞危险因素下的最佳抗凝强度。结果:本研究共纳入3 176例患者,其中3 017例获随访,随访率95%,159例失访(出院后未获得随访资料),失访率5%,3 017例患者共随访12 746.08人年(平均每位患者随访4.22年)。随访期间,共185例患者发生不良事件,约为1.45/100人年(95%可信区间[CI],1.22-1.64)。其中54名患者发生严重出血事件(0.42/100人年,95%CI,0.31-0.53),131名患者发生血栓栓塞事件(1.03/100人年,95%CI,0.85-1.21)。总体双叶机械瓣瓣膜置换术患者的最佳抗凝强度范围,INR位于1.5-2.5之间。主动脉瓣置换术(AVR)且无血栓栓塞危险因素的患者,最佳INR范围为1.5-2.0;二尖瓣置换术(MVR)且无血栓栓塞危险因素的患者,最佳INR为1.5-2.0,而合并血栓栓塞危险因素的患者,最佳INR为2.0-2.5;主动脉瓣和二尖瓣置换术(DVR)的患者,其最佳抗凝强度与单纯MVR相同。涉及三尖瓣置换(TVR)的患者,本研究不能有效评估其最佳抗凝强度,基于三尖瓣位具有较高血栓形成性,其最佳抗凝强度的最低标准为 INR 2.0-2.5。结论:中国人双叶机瓣瓣膜置换的患者,最佳的抗凝水平的INR目标范围为1.5-2.5(2.0)。但个体最佳抗凝强度应根据不同瓣膜位置及有无血栓栓塞危险因素进行适当调整。第二章:心脏机械瓣膜置换术后抗凝过度终止抗凝的方法学研究目的:通过比较口服维生素K1与口服安慰剂,以及比较口服维生素K1与静脉或肌肉注射维生素K1治疗心脏机械瓣膜置换术后口服华法林抗凝过度(INR 4.0-10.0)且无临床出血症状者的疗效与安全性,评估此类患者的最佳治疗方案。方法:本研究为单中心前瞻性的随机对照试验。研究共分为三组随机对照,分别是:口服安慰剂与口服维生素K1对照,维生素K1口服和静脉注射途径的对照以及口服和肌肉注射途径的对照。研究纳入2018年1月至2021年1月我院门诊和住院的双叶机械瓣膜置换后口服华法林抗凝治疗INR 4.0-10.0之间且无临床出血症状的患者。按照不同试验分组进行随机分组试验,维生素K1或安慰剂的给药剂量为2.5mg。所有受试者给药4小时、8小时、12小时、24小时监测一次INR值,之后每24小时监测一次INR值至INR达到目标水平(1.5-2.5),调整药物后3天,7天,1月、2月、3月随访并检测INR值。所有患者随访满3月后完成心脏彩超检查。同时,口服安慰剂组和不同途径维生素K1给药组各抽取10例行凝血因子检测。研究期间记录所有患者维生素K1不良反应事件,以及血栓栓塞事件和出血事件。结果:本研究从2018年1月至2021年1月共筛选抗凝过度的患者236例,其中16例被排除,剩余220例患者参与研究,完整随访210例,随访率达95%。所有患者均未出现华法林抵抗和维生素K1过敏反应。1.口服安慰剂和口服维生素K1的对照:口服安慰剂组和口服维生素K1组各纳入患者40例。用药后,安慰剂组抗凝过度纠正(INR≤2.5)所需时间明显高于维生素K1组(3.45±1.34 vs 1.15±0.36 天,P<0.001)。服药 24 小时,维生素K1组INR值达到抗凝目标范围(INR 1.5-2.5)的百分比明显高于安慰剂组(72.5%vs0%,P<0.001)。用药后8-72小时,安慰剂组INR平均值均明显高于维生素K1组(P<0.05)。此外,与安慰剂组相比,维生素K1组患者的总出血发生率显著降低(P=0.025;OR 0.33,95%CI 0.11-0.88)。2.口服维生素K1与静脉注射维生素K1的对照:口服组纳入患者38例,静脉组纳入患者37例。用药后24小时,口服组INR达标率(INR 1.5-2.5)显著高于静脉组(71.95%vs 35.14%,P<0.001),而静脉组纠正过度(INR<1.5)比例显著高于口服组(56.76%vs 13.16%,P<0.001)。维生素K1用药后12小时内,静脉组纠正INR(INR≤2.5)的百分比明显高于口服组(P<0.05)。用药后12-72小时,静脉组纠正过度(INR<1.5)的百分比明显高于口服组(P<0.05)。用药后4-72小时,静脉组的INR均值均明显低于口服组(P<0.05)。两组血栓栓塞事件和出血事件无统计学差异。3.口服维生素K1与肌肉注射维生素K1的对照:口服组纳入患者33例,肌注组纳入患者32例。口服组和肌注组停药天数分别为1.15±0.44天和2.31±0.90天(P<0.001)。服药后24小时,口服组INR纠正达标率(INR 1.5-2.5)明显高于肌注组(78.79%vs 18.75%,<0.001)。相反,肌注组用药24小时抗凝过度 INR>2.5 的有 26 例(81.25%,95%CI 64-93),INR>4.0 的有10例(31.25%,95%CI 16-50),均显著高于口服组(P<0.001)。维生素K1用药后12-48小时,口服组纠正INR(INR≤2.5)的百分比均明显高于肌注组(P<0.05)。肌注组总出血率高于口服组,但两组血栓栓塞事件无统计学差异。4.凝血因子监测:安慰剂组,维生素K1口服、静脉以及肌注组患者凝血因子的变化和INR值的变化基本一致。用药24小时后,凝血因子Ⅱ、Ⅶ、Ⅸ、X的水平高低排序为静脉组>口服组>肌注组>安慰剂组。结论:1.中国人心脏机械瓣膜置换术后使用华法林抗凝治疗的患者,出现抗凝过度INR4.0-10.0之间且临床无出血症状时,应积极予以维生素K1治疗。2.低剂量维生素K1对抗凝过度纠正抗凝的治疗是有效的,无华法林抵抗等不良结局,但安全性尚需进一步大样本的研究予以论证。3.本研究从临床治疗效果的角度证明了维生素K1的三种给药途径的疗效差异:静脉给药起效最快,疗效肯定;口服起效仅次于静脉,且24小时后可达到与静脉途径相似的疗效,在非紧急情况下更安全;肌注起效最慢,疗效最差。第三章:心脏机械瓣膜置换术后抗凝过度的遗传因素研究目的:通过比较心脏机械瓣膜置换术后,口服华法林抗凝过度和抗凝稳定患者之间的遗传因素差异,为华法林抗凝过度患者的合理用药提供遗传学基础。方法:本研究为回顾性非随机对照试验。研究根据INR测定结果和稳定情况分为抗凝过度组和抗凝稳定组,抗凝过度组纳入我院门诊及住院机械瓣膜置换术后口服华法林治疗,不明原因抗凝过度INR大于4.0的汉族患者;抗凝稳定组纳入我院门诊机械瓣膜置换术后长期口服华法林抗凝治疗(抗凝治疗大于6月),连续两次监测INR稳定于1.5-2.5之间,且3个月内未调整过华法林剂量的汉族患者。采用限制性片段长度多态性聚合酶链反应(PCR-RFLP)技术检测VKORC1 1639G>A、CYP2C9*2以及CYP2C9*3基因多态位点,比较两组患者上述基因多态位点突变的差异以及两组患者基因多态位点与华法林平均每日维持剂量的差异。抗凝过度组患者需随访至患者达到抗凝稳定状态,并记录此类患者抗凝稳定后的华法林剂量。结果:研究自2015年7月至2020年12月,通过纳入和排除标准,抗凝过度组和抗凝稳定组分别纳入128例和130例患者进行研究。两组患者身高、体重和体表面积与华法林每日维持剂量间存在明显的正相关关系。但通过线性回归分析可知,抗凝过度组线性回归方程式的拟合度(R2)和直线斜率均低于抗凝稳定组。抗凝过度组CYP2C9基因的野生型患者比例明显低于抗凝稳定组(72.66%vs 90.00%,OR 0.30,95%CI 0.15-0.58,P<0.001),而 CYP2C9*3 杂合突变携带者却明显高于抗凝稳定组(24.22%vs 9.23%,OR 3.01,95%CI 1.49-6.09,P<0.001)。VKORC1-1639位点的突变与高剂量华法林相关,而CYP2C9位点的突变与低剂量华法林相关。由于抗凝过度组中同时携带VKORC1-1639 AA和CYP2C9*1*3的患者比例显著高于抗凝稳定组(21.88%vs 9.23%,P=0.02),而抗凝过度组同时携带VKORC1-1639 AA和CYP2C9*1*1的比例则明显低于抗凝稳定组(50.78%vs 90.00%,P<0.001),所以抗凝过度组VKORC1-1639位点为野生型(AA)的患者华法林每日维持剂量显著低于抗凝稳定组(2.65 ±0.92 mg/d,3.01±1.22 mg/d,P=0.02)。结论:1.中国人机械瓣膜置换术后服用华法林抗凝过度的汉族患者,CYP2C9*3基因多态位点的突变率明显高于抗凝稳定的患者,进而导致抗凝过度人群的华法林平均每日需求量减低,同时也弱化了非遗传因素对华法林剂量的影响。2.本研究为回顾性的研究,发现了抗凝过度人群的遗传学基础,为抗凝过度患者的精准化治疗提供新的思路。同时,为下一步前瞻性的验证试验和基因指导抗凝过度患者的华法林剂量调整的随机对照试验提供依据。更多还原

【Abstract】 BackgroundAll patients that receive such mechanical valves generally need to be treated via anticoagulant therapy for the remainder of their lives,with vitamin K antagonists(VKA)being the only effective orally deliverable options,among which warfarin is the most frequently used.It is necessary to develop a reasonable individualized anticoagulant therapy for warfarin,due to its safety range is narrow,and the factors affecting the anticoagulantion of warfarin is numerous.The oral anticoagulant therapy intensity of warfarin including the following three aspects:the optimal INR,subtherapeutic INR and excessive anticoagulation.There is no guideline for warfarin anticoagulantion in China and the only expert consensus is based on Western guidelines.It is inappropriate to apply Western anticoagulant guidelines to Chinese people,because the optimal INR of Chinese people is lower than that of Westerner.It remains controversial as to what this optimal intensity level and best therapeutic treatment of excessive anticoagulation is among Chinese patients with bileaflet mechanical valves.Chapter 1 Optimal oral anticoagulant therapy in patients with mechanical heart valvesObjective:By reviewing the actual anticoagulant strength data and calculating the incidence of adverse events in different ranges of anticoagulant strength,we sought to better determine what the ideal oral anticoagulant therapy intensity is in a Chinese population of patients implanted with mechanical valves.Methods:This is a prospective follow-up,retrospective data study.We observed the outcomes of eligible enrolled Chinese patients that had been implanted with St.Jude Medical prosthetic valves and who had been treated at our institution from 2013 onwards.All enrolled patients were followed from one to six years by outpatient visits or telephone interviews following discharge.Prothrombin time was monitored a minimum of once every 56 days,given that we were unable to assume that linear changes would occur between temporally distant follow-up time points.Study endpoints included instances of thromboembolism and major hemorrhage.We calculated the international normalized ratio(INR)-specific incidence of such adverse events as a means of determining optimal anticoagulant therapeutic intensity in Han patients with these bileaflet mechanical valve prostheses.We further assessed optimal VKA dosing intensity in patients as a function of patient valve site and thromboembolism risk via separating patients according to their valve sites or risk level(high or low)and then conducting subgroup analyses.Results:In total this study enrolled 3176 patients,of whom 3017 were followed for 12,746.08 total person-years(4.22 mean years per patient),while the remaining 159 patients(5%)were lost during follow-up.A total of 185 patients suffered from adverse events,with a roughly 1.45%annual incidence rate of such adverse events(95%confidence interval[CI],1.22-1.64).Of these,54 patients suffered from episodes of major bleeding(0.42%per patient-year,95%CI,0.31-0.53),while 131 suffered from thromboembolism(1.03%per patient-year,95%CI,0.85-1.21).Based on these analyses,we determined that the ideal low-intensity anticoagulant treatment regimen for patients with bileaflet mechanical heart valve prostheses was an INR between 1.5 and 2.5.In those patients that had undergone aortic valve replacement(AVR),the optimal INR was between 1.5 and 2.0 regardless of any thromboembolism risk factors in these patients.In contrast,in patients that had undergone mitral valve replacement(MVR)who were not at risk for thromboembolism,the optimal INR was between 1.5 and 2.0,whereas in those at risk of thromboembolism the optimal INR was between 2.0 and 2.5.The ideal anticoagulant intensity in patients undergoing both AVR and MVR(DVR)was the same as in patients undergoing MVR alone.Due to the small number of patients with TVR,the ideal intensity of anticoagulation has not been determined in our study.However,according to the particularity of tricuspid valve location,it is more prone to thrombosis.We recommend that the intensity of anticoagulation should reach INR 2.0-2.5 at least.Conclusion:In order to achieve optimal outcomes by maintaining effective anticoagulant dosing while entailing minimal risk in Chinese patients that have undergone a bileaflet mechanical valve replacement,the target INR range should be between 1.5 and 2.5,with the final intensity of this anticoagulant regimen being determined based on a given patient’s risk of thromboembolism.Chapter 2 The treatment strategies for excessive anticoagulation in patients with mechanical heart valvesObjective:This trial aimed to determine the safety and efficacy of using low-dose vitamin K1(different administration routes)or Placebo to lower international normalized ratio(INR)values into the target range in a cohort of Chinese patients with mechanical heart valves.We sought to determine what the ideal treatment strategies for excessive anticoagulation are in a Chinese population of patients implanted with mechanical valves.Methods:This study was a single-center prospective randomized controlled trial which was divided into three randomized controls.The three parts are as follows:oral Vitamin K1 vs placebo,oral vs intravenous Vitamin K1,oral vs intramuscular Vitamin K1.Patients receiving warfarin after mechanical valve replacement in our institution,who had an INR value between 4.0 and 10.0,were enrolled in the study between January 2018 and January 2021.Randomized controlled trials were conducted according to the experimental scheme.The dose of vitamin K1 or placebo was 2.5mg.The INR values were monitored for all subjects at 4,8,12 and 24h after administration.The INR values were then monitored every 24 hours until the target range was reached.All subjects were followed up and INR values were measured at 3 days,7 days,1 month,2 months and 3 months after warfarin adjustment.Vitamin K-dependent coagulation factors were monitored for ten patients who were selected from the oral,intravenous,intramuscular injection of Vitamin K groups and placebo group.Thromboembolism,bleeding,Vitamin K1 anaphylaxis,warfarin resistance and other adverse events were recorded in all patients over the period studied.Results:The patients of 236 were screened in the study between in January 2018 and January 2021,of whom 220 participated in the study.The patients of 10(5%)were lost during follow-up.All patients did not have vitamin K1 hypersensitivity reaction and warfarin resistance.1.Oral Vitamin K vs Placebo:Forty patients were included in the placebo group and the vitamin K group respectively.The placebo group took significantly longer for normalisation of their INR(INR≤2.5)than the vitamin K group(3.45±1.34 vs 1.15±0.36 day,P<0.001)after treatment.The percentage of patients who received vitamin K1 had INR values of 1.5-2.5 was significantly higher than those who received placebo the day after the study drug(72.5%vs 0%,P<0.001).The placebo group exhibited significantly higher average of values of INR relative to the vitamin K1 group on first 8-72 hours following treatment(P<0.05).When all 80 patients were incorporated into the safety analysis,we observed a significantly reduced bleeding incidence in patients of vitamin K group relative to the placebo group(p=0.025;OR 0.33,95%CI 0.11-0.88).2.Oral vs Intravenous Vitamin K:38 patients were included in the oral administration of Vitamin K1 group and 37 patients in the intravenous Vitamin K1 administration.The percentage of patients who received oral vitamin K1 had INR values of 1.5-2.5 was significantly higher than those who received intravenous Vitamin K1 on 24 hours after taking the drug(71.95%vs 35.14%,P<0.001).56.76%of the patients had INR values less than 1.5 after intravenous administration and 13.16%after oral administration(P<0.001).The percentage of patients with the values of INR≤2.5 within 12 hours after treatment was considerably higher for the intravenous group relative to oral group(P<0.05).At the same time,The percentage of patients with the values of INR<1.5 on 12-72 hours after treatment was considerably higher for the intravenous group relative to oral group.The oral group exhibited significantly higher average of values of INR relative to the intravenous group on 4-72 hour following treatment(P<0.05).There was no significant difference in thromboembolic events and bleeding events between the two groups.3.Oral vs Intramuscular Vitamin K:33 patients were included in the oral administration of Vitamin K1 group and 32 patients in the intramuscular Vitamin K1 administration.The duration of warfarin withdrawal in the oral vitamin K1 group and the intramuscular injection vitamin K1 group were 1.15±0.44 day and 2.31±0.90 day respectively(P<0.001).The percentage of patients who received oral vitamin K1 had INR values of 1.5-2.5 was significantly higher than those who received Intramuscular Vitamin K1 on 24 hours after administration of Vitamin K1(78.79%vs 18.75%,<0.001).However,At 24 hours,26/32(81.25%)patients had an INR greater than 2.5 and 10(31.25%)patients failure to respond to Vitamin K1(INR>4.0 at 24 hours)in the Intramuscular Vitamin K1 group.At 12 to 48 hours after taking vitamin Kl,the percentage of patients in the oral administration of Vitamin K group had INR values less than or equal 2.5 was significantly higher than those in the Intramuscular Vitamin K group.The bleeding events significantly higher in the intramuscular injection vitamin K1 group than and the oral vitamin K1 group.But there was no significant difference in thromboembolic events between the two groups.4.Coagulation factor monitoring:The trends of coagulation factor had strong correlation with the INR change.At 24 hours after treatment,the order of clotting factor levels is as follows:Intravenous Vitamin K1>Oral Vitamin K1>Intramuscular Vitamin K1>Oral Placebo.Conclusion:1.Chinese patients treated with warfarin anticoagulation after mechanical valve replacement with INR values of 4.0 to 10.0 without bleeding should be actively given vitamin K1 treatment.2.Low dose vitamin K1 is effective to reverse Excessive Anticoagulation;however,the safety of drug application needs to expand sample size in future studies.3.Intravenous administration has the fastest effect and definite curative effect,followed by oral,and the effect of oral administration is similar to that of intravenous administration after 24 hours of treatment.Intramuscular vitamin K was the least effective owing to the irregular and unpredictable absorption.Chapter 3 Genetic factors of excessive anticoagulation in patients with mechanical valve replacementObjective:To compared the differences of genetic factors between patients of excessive anticoagulation and stable anticoagulation,we sought to determine the genetic underpinnings for rational medicine use of warfarin in Chinese patients with excessive anticoagulation.Methods:This study was a retrospective,non-randomized controlled trial,which was divided into excessive anticoagulation and stable anticoagulation group.Patients receiving warfarin after mechanical valve replacement in our institution,who had an INR value between 4.0 and 10.0 of unknown cause,were enrolled in the excessive anticoagulation group.Patients with mechanical heart valves,who take orally warfarin anticoagulant therapy got two times stable INR(INR1.5-2.5)at least and with no need to adjust VKA dosing at least 3 months were recruited in stable anticoagulation group.Polymerase Chain Reaction-Restricted Fragment Length Polymorphisms(PCR-RFLP)was used to test the polymorphisms of VKORCI and CYP2C9genes.The mutations of the above polymorphism sites,the differences of gene and warfarin dose were compared between the two groups.Patients in the excessive anticoagulation group were followed up until the patients reached a stable anticoagulantion.Warfarin dose was recorded after anticoagulant stabilization in these patients.Results:According to inclusion and exclusion criteria,the patients of 128 and 130 were enrolled in the excessive anticoagulation group and the stable anticoagulantion group,respectively,from July 2015 to December 2020.There was a significant positive correlation between height,weight,BSA and daily warfarin maintenance dose in both groups.However,the linear regression analysis showed that the fitting degree(R2)and linear slope of the linear regression equation in the excessive anticoagulation group were lower than those in the stable anticoagulantion group.The proportion of wild-type patients with CYP2C9 gene in the excessive anticoagulation group was significantly lower than that in the stable anticoagulantion group(72.66%vs 90.00%,OR 0.30,95%CI 0.15-0.58,P<0.001).And CYP2C9*3 heterozygous mutation in the excessive anticoagulation group were significantly higher than those in the stable anticoagulantion group(24.22%vs 9.23%,OR 3.01,95%CI 1.49-6.09,P<0.001).Percentage of patients carrying VKORC1-1639 AA and CYP2C9*1*3 in the excessive anticoagulation group was significantly higher than that in the stable anticoagulantion group(21.88%vs 9.23%,P=0.02).In contrast,Percentage of patients carrying VKORC1-1639 A A and CYP2C9*1*1 in the excessive anticoagulation group was significantly lower than that in the stable anticoagulantion group(50.78%vs 90.00%,P<0.001).Mutations at VKORC1-1639 were associated with high doses of warfarin.Mutations at CYP2C9 were associated with low doses of warfarin.The mean daily warfarin dosage of patients with wild-type patients with VKORC1-1639 in the excessive anticoagulation group was significantly lower than that in the stable anticoagulantion group(2.65±0.92 mg/d,3.01±1.22 mg/d,P=0.02).Conclusion:1.The mutation rate of polymorphic sites of CYP2C9*3 in Chinese Han patients with excessive anticoagulation was significantly higher than those with stable anticoagulantion,in turn,lead to the average daily requirement for warfarin in the excessive anticoagulation population is reduced.Finally,the effect of non-genetic factors on warfarin dose was weakened.2.This study found the genetic basis of excessive anticoagulation,which provides a new idea for the precise treatment of patients with excessive anticoagulation.At the same time,it provides the basis for future prospective validation test and Randomized,Controlled trial of Genotype-guided Warfarin Prescribing for patients with excessive anticoagulation.更多还原