非维生素K依赖的抗凝药对凝血酶生成的影响及维生素K依赖的口服抗凝药起始应用过程中凝血酶生成能力的变化趋势

发布时间：2018-04-24 14:34

本文选题：达比加群 + 利伐沙班　；参考：《北京协和医学院》2016年博士论文

【摘要】：第一部分达比加群和利伐沙班对组织因子诱导的凝血酶生成和血小板聚集功能的影响目的：达比加群酯和利伐沙班是新近应用于临床的非维生素K依赖的直接凝血酶和直接因子Xa抑制剂。临床研究已经证明二者的疗效不劣于甚至优于传统的抗凝药物华法林。由于作用靶点的不同,二者抗凝效应必然存在差异。本研究通过体外血浆凝血酶生成(TG)和全血血小板聚集率(PAgR)试验评价达比加群和利伐沙班对组织因子诱导的凝血过程的影响,并探讨凝血酶调节蛋白(TM)在抗凝药物环境下对TG的调节作用。方法：枸橼酸钠抗凝管采集健康受试者外周静脉全血,按以下三个方面处理：①分离乏血小板血浆(PPP),体外给予递增浓度梯度(0,25,100,400,800 nmol/l)的达比加群和利伐沙班预处理,应用校正的自动凝血酶生成图(CAT)法检测TG参数,包括延迟时间(Lagtime)、内源性凝血酶生成能力(ETP)、峰值(Peak)和达峰时间(Ttpeak);②枸橼酸钠抗凝全血在体外给予上述浓度梯度的达比加群和利伐沙班预处理,动态血小板计数法检测全血PAgR;③在①基础上加用等量的重组人凝血酶调节蛋白(rhs-TM,10 nmol/l),观察TG变化。结果：①达比加群(0-800 nmol/l)浓度依赖性的延长血浆Lagtime和Ttpeak等时间参数。低浓度的达比加群(25-100 nmol/l)不降低血浆ETP(1737.6-1904.7 nmol-min)和Peak (337.7-364.6 nmol),高浓度的达比加群(100-800 nmol/L)浓度依赖性降低血浆ETP和Peak。利伐沙班(0-800nmol/l)延长Lagtime和Ttpeak及降低ETP和Peak均呈浓度依赖性的。以ETP计算,达比加群和利伐沙班的半数最大抑制浓度(IC50)分别为678.1±1.4 nmol/l和460.1±1.4 nmol/l,二者比值约1.47倍。②在空白对照组,组织因子可诱导78±5%的全血PAgR;达比加群和利伐沙班(0-800nmol/l)均可浓度依赖性的抑制组织因子诱导的PAgR, IC50分别为119.5±1.5和77.5±1.6nmol/l,二者比值约1.54倍。③rhs-TM不影响Lagtime和Ttpeak,但显著降低ETP和Peak(分别下降22.4%和13.3%)。在rhs-TM环境下,低浓度的达比加群(25 nmol/l)有增加ETP(1568.97±117.99 vs 1478.65 ±73.77 nmol-min)和Peak(330.82±17.56 vs 299.46 ±6.35 nmol/l)的趋势(P0.05)。不同的是,低浓度利伐沙班(25 nmol/)即可显著降低ETP (748.42±368.97 vs 1478.65 ±73.77 nmol-min)和Peak(106.53±55.46 vs 299.46 ±6.35 nmol/l)(P均0.01)。以ETP计算,达比加群和利伐沙班的IC50分别为605.5±1.9 nmol/l和24.7±1.1 nmol/l,二者比值约24.5倍。结论：达比加群浓度依赖性的延迟TG的起始期,低浓度的达比加群(≤100nmol/l)对TG的扩增期无影响。利伐沙班浓度依赖性的延迟TG的起始期、抑制TG扩增期。利伐沙班和达比加群能浓度依赖性的抑制组织因子诱导的全血PAgR。利伐沙班对血浆TG和全血PAgR的抑制效应强于达比加群。rhs-TM能增强利伐沙班的抗凝效应,对达比加群的抗凝效应的影响较小。第二部分心房颤动患者应用华法林起始抗凝治疗过程中血浆内源性凝血酶生成能力变化趋势及临床意义目的：具有高危血栓风险的心房颤动患者积极抗凝治疗可改善临床预后。间接证据表明华法林起始应用过程中可能出现反常高凝和增加血栓事件发生。本研究评价心房颤动患者应用华法林起始抗凝阶段血浆内源性凝血酶生成能力(ETP)的变化趋势,探讨这一变化趋势的临床意义。方法：前瞻性收集具有抗凝适应症但近2周内未行抗凝治疗的心房颤动患者39例,按起始抗凝方案中是否联合应用低分子肝素(1OOAxaIU/kg)分成2组：A组：华法林组,26例；B组：联合治疗组,13例。A组按华法林给药剂量分为三个亚组：A1：1.5mg qd,7例；A2:3mg qd,11例；A3:6mg qd×2d,然后3mg qd,8例。收集患者人口学特征、病史资料、基线血常规、D二聚体和纤维蛋白原水平。枸橼酸钠抗凝管采集患者基线和用药后连续3天的静脉全血。采用校正的自动凝血酶生成图(CAT)法检测ETP。同时间点检测国际标准化比值(INR)、活化部分凝血活酶时间(APTT)、蛋白C(PC)和蛋白S(PS)等。多元线性回归分析ETP与其它因素的关系。结果：除了糖尿病病史两组之间存在显著差异外(3.8% vs 30.8%,P=0.035),其它基线特征、基线INR、APTT、PC、PS和ETP等均未见显著差异。2组患者INR动态比较无显著差异(累计增幅33.8%vs 34.8%, P=0.947); APTT在华法林组表现为逐步升高的过程；在联合治疗组第1天即显著升高(增幅14.5%vs 3%,P=0.026)并维持这一水平,至第3天两组APTT无显著差异(增幅13.5%vs 15.84%,P=0.628)；两组PC和PS活性均呈现缓慢下降趋势,PC/PS累计降幅均无显著差异(分别为-28.6%vs-23.9%,-18.6%vs-14.5%：P值分别为0.505和0.565),其中第2天华法林组PC日降幅显著高于联合治疗组(-14.7%vs -7.0%,P=0.033)。联合治疗组ETP随时间推移直线下降；华法林组ETP的降幅在第2天呈小幅波动,两组患者3天ETP累计降幅无显著差异(-30.5% vs -36.8%,P=0.273)。多元线性回归分析提示基线ETP与红细胞(RBC)计数(p=257.6,P=0.001)和INR独立相关(p=-1425.8,P=0.001)；第1天和第2天ETP的日降幅与同时段INR变化幅度独立负相关(分别β=-0.946,-0.231；P=0.002,0.004),3天累计ETP降幅与同时段PC降幅独立负相关(p=-0.378,P=0.002)。亚组分析发现,华法林对PC、PS和ETP的抑制均呈现剂量依赖性。A1组3天ETP累计降幅显著低于A2和A3组(分别为-20.4%,-34.1%和-34.3%)；A1和A2组ETP在第3天出现最大降幅(分别为-8.7%和-21.7%)；A3组ETP在第1天的降幅最大(-17.5%)。结论：小剂量的华法林起始抗凝治疗过程中ETP最大降幅出现在第3天,大剂量的华法林在第1天即可获得较显著的ETP降幅。华法林对ETP、PC和PS活性的影响均呈现剂量依赖性。心房颤动患者应用华法林起始抗凝过程中ETP的日降幅呈现小幅波动,该现象可能通过联合低分子肝素治疗获得改善。RBC计数和INR值影响基线ETP水平；而华法林起始给药过程中INR值和PC活性的变化与ETP的变化相关。
[Abstract]:The first part of the effect of dabiga group and LEV Shaaban on tissue factor induced thrombin formation and platelet aggregation purpose: dabig and LEV Shaaban are newly used direct thrombin and direct factor Xa inhibitors for clinical non vitamin K dependence. Clinical studies have shown that the efficacy of the two is not inferior to even superior. The anticoagulant effect of the traditional anticoagulant drug, warfarin, is necessarily different from the two anticoagulant effects. In this study, the effects of dabiga group and LEV Shaaban on the coagulation process induced by tissue factors were evaluated by plasma thrombin formation (TG) and blood platelet aggregation (PAgR) test in vitro, and the thrombin regulatory protein (TM) was discussed. The regulation of TG under the anticoagulant environment. Methods: the sodium citrate anticoagulant tube was used to collect the peripheral venous blood of the healthy subjects. According to the following three aspects: (1) the isolated platelet plasma (PPP) was separated, and the dabiga group and LEV Shaaban pretreated with the increasing concentration gradient (0,25100400800 nmol/l) were pretreated in vitro, and the corrected automatic coagulation was applied. TG parameters were detected by CAT, including delayed time (Lagtime), endogenous thrombin generation capacity (ETP), peak (Peak) and peak time (Ttpeak); (2) sodium citrate anticoagulant whole blood was given the above concentration gradient of dabiga group and ribeasta preconditioning in vitro, dynamic platelet counting method was used to detect PAgR in whole blood; (3) on the basis of 1 The same amount of recombinant human thrombin regulated protein (rhs-TM, 10 nmol/l) was added to observe the changes in TG. Results: (1) dabiga group (0-800 nmol/l) concentration dependent prolongation of plasma Lagtime and Ttpeak time parameters. Low concentration dabia group (25-100 nmol/l) did not reduce plasma ETP (1737.6-1904.7 nmol-min) and Peak (337.7-364.6), high concentration Dabiga group (100-800 nmol/L) concentration dependent reduced plasma ETP and Peak. Lev Shaaban (0-800nmol/l) prolonged Lagtime and Ttpeak and reduced ETP and Peak in a concentration dependent manner. In ETP, the maximum inhibitory concentration of dabiga group and LEV Shaaban (IC50) is 678.1 + 1.4 nmol/l and 460.1 + 1.4 nmol/l, and the two ratio is about 1.47 times. (2) in the blank control group, the tissue factor could induce 78 + 5% of the whole blood PAgR; dabiga group and LEV Shaaban (0-800nmol/l) could inhibit the concentration dependent tissue factor induced PAgR, IC50 was 119.5 + 1.5 and 77.5 + 1.6nmol/l respectively, the ratio of two was about 1.54 times. (3) rhs-TM did not sound Lagtime and Ttpeak, but significantly decreased ETP and Peak (respectively decreased. 22.4% and 13.3%). Under the rhs-TM environment, the low concentration dabiga group (25 nmol/l) has a trend of increasing ETP (1568.97 + 117.99 vs 1478.65 + 73.77 nmol-min) and Peak (330.82 + 17.56 vs 299.46 + 6.35 nmol/l). And Peak (106.53 + 55.46 vs 299.46 + 6.35 nmol/l) (P 0.01). The IC50 of dabiga group and LEV Shaaban's IC50 were 605.5 + 1.9 nmol/l and 24.7 + 1.1 nmol/l respectively. The ratio of two was about 24.5 times respectively. Conclusion: dabiga group concentration dependent delayed TG initiation period and low concentration Dabi group (< < < 100nmol/l) have no effect on TG amplification period. The starting period of Lev Shaaban concentration dependent delayed TG inhibited the TG amplification period. The inhibitory effect of Lev Shaaban and dabiga group on the inhibitory effect of PAgR. Lev Shaaban on plasma TG and whole blood PAgR was stronger than that of dabiga group.Rhs-TM to enhance the anticoagulant effect of Lev Shaaban, and the anticoagulant effect on dabiga group The change trend and clinical significance of plasma endogenous thrombin formation in second parts of atrial fibrillation patients during the initial anticoagulant treatment of atrial fibrillation: positive anticoagulant therapy with high risk of thrombus risk in patients with atrial fibrillation can improve clinical prognosis. Abnormal hypercoagulability and increased thrombotic events occurred. This study evaluated the changes in plasma endogenous thrombin formation (ETP) in warfarin initial anticoagulant stage in patients with atrial fibrillation, and explored the clinical significance of this trend. Method: prospective collection of atrial fibrillation with anticoagulant therapy in the near 2 weeks without anticoagulant therapy 39 patients were divided into 2 groups according to the combined application of low molecular weight heparin (1OOAxaIU/kg) in the initial anticoagulant regimen: group A: warfarin group, 26 cases; group B: combined treatment group; 13 cases of group.A were divided into three subgroups according to the dosage of warfarin: A1:1.5mg QD, 7 cases; A2:3mg QD, 11 cases; A3:6mg QD x 2D, then 3mg 8. Signs, medical history data, baseline blood routine, D two polymer and fibrinogen level. The sodium citrate anticoagulant tube collected the patient's baseline and 3 days of continuous venous blood after 3 days of medication. The revised automatic thrombin generation chart (CAT) was used to detect the international normalized ratio (APTT), the activated partial thromboplastin time (APTT), and the protein C (PC) at the same time point. Protein S (PS) and so on. Multivariate linear regression analysis of the relationship between ETP and other factors. Results: there were significant differences between the two groups (3.8% vs 30.8%, P=0.035), other baseline features, baseline INR, APTT, PC, PS and ETP, and no significant difference in INR dynamics (cumulative increase 34.8%). APTT in the warfarin group showed a gradual increase in the performance of the warfarin group, which was significantly increased (14.5%vs 3%, P=0.026) and maintained this level in the first days of the combined treatment group, and there was no significant difference between the two groups (13.5%vs 15.84%, P=0.628) in the third day group, and the two groups of PC and PS activity showed a slow decline trend, and there was no significant difference in the cumulative decrease of PC/PS. The value of -18.6%vs-14.5%:P was 0.505 and 0.565 respectively, and the decrease of PC day in second days was significantly higher than that in the combined treatment group (-14.7%vs -7.0%, P=0.033). The ETP in the combined treatment group decreased linearly with time, and the decrease of ETP in the warfarin group was slightly fluctuating in second days, and there was no significant difference between the two groups of patients at 3 days. -30.5% vs -36.8%, P=0.273). Multivariate linear regression analysis suggested that baseline ETP and RBC count (RBC) count (p=257.6, P=0.001) and INR independent correlation (p=-1425.8, P=0.001); first days and second days, the daily decline of ETP is independent of the amplitude of simultaneous variation (beta =, respectively), and 3 days' cumulative decline and simultaneous decline Independent negative correlation (p=-0.378, P=0.002). The subgroup analysis found that Hua Falin's inhibitory effect on PC, PS and ETP was significantly lower in the dose dependent.A1 group than the A2 and A3 groups at 3 days (-20.4%, -34.1% and -34.3%). 5%). Conclusion: the maximum ETP decline occurred during the small dose of Hua Falin initial anticoagulant therapy in third days, the large dose of Hua Falin could obtain a significant ETP decline in first days. The effect of Hua Falin on the activity of ETP, PC and PS showed a dose dependence. The decrease of the daily decrease of ETP during the application of the initial anticoagulant process in the patients with atrial fibrillation was small. Amplitude fluctuation, this phenomenon may improve the.RBC count and INR value by combined low molecular weight heparin therapy, and the changes of INR and PC activity during the initial administration of warfarin are related to the changes of ETP.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R541.75

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