| Part I:An in-vitro Evaluation of Dabigatran and Rivaroxaban on Tissue Factor-Induced Thrombin Generation and Platelet AggregationObjective:Dabigatran and rivaroxaban are non-vitamin K dependent oral anticoagulants that specifically inhibit thrombin and factor Xa, respectively. Clinical studies have proved that the efficacies of dabigatran and rivaroxaban were non-inferior or superior to traditional vitamin K-dependent agent warfarin. However, there must be differences in details between dabigatran and rivaroxaban due to the different anticoagulant targets in the coagulation cascade. The aim of this study is to investigate the in-vitro impact of dabigatran and rivaroxaban on thrombin generation (TG) and platelet aggregation derived via extrinsic pathway, and evaluate the regulation of thrombomodulin (TM) on TG.Methods:Citrated human blood samples were obtained from health adults and tested according to the following procedures. ①Platelet-poor plasma(PPP) was isolated and incubated with vehicle or increasing concentrations (25,100,400,800 nmol/l) of dabigatran and rivaroxaban. TG was detected by calibrated automated thrombogram assay (CAT), and the parameters of Lagtime, endogenous thrombin potential (ETP), Peak and time to peak (Ttpeak) were recorded. ② Whole blood was pretreated by vehicle or increasing concentrations (25,100,400,800 nmol/l) of dabigatran and rivaroxaban, and the platelet aggregation ratio (PAgR) was tested by a kinetic counting method. ③ PPP was co-incubated with vehicle or recombinant human soluble TM (rhs-TM,10 nmol/l), and then pretreated with vehicle or increasing concentrations (25,100,400,800 nmol/l) of dabigatran and rivaroxaban, and detected by CAT.Results:①Dabigatran (0-800 nmol/l) concentration-dependently affected kinetic parameters with prolonged Lagtime and Ttpeak. Dabigatran did not reduce the quantitative parameters with similar ETP (1737.6-1904.7 nmol min) and Peak (337.7-364.6 nmol) in lower concentrations (25-100 nmol/l), but exhibited a concentration-dependently inhibition in higher concentrations (100-800 nmol/l). Rivaroxaban (0-800 nmol/l) concentration-dependently affected both kinetic and quantitative parameters. The IC50s of dabigatran and rivaroxaban on ETP were 678.1±1.4 nmol/l and 460.1±1.4 nmol/l, respectively (ratio:1.47). ② Tissue factor produced a PAgR of 78±5% in the vehicle-treated whole blood. Both dabigatran and rivaroxaban concentration-dependently inhibited PAgR with the IC50s of 119.5 ±1.5 and 77.5±1.6nmol/l, respectively (ratio:1.54). ③ Rhs-TM lowered down ETP and Peak (22.4% and 13.3%, respectively) in vehicle-treated PPP, but did not prolong Lagtime and Ttpeak. When co-incubated with rhs-TM, dabigatran at 25 nmol/l induced an increasing tendency in ETP (1568.97±117.99 vs 1478.65±73.77 nmol-min) and Peak (330.82±17.56 vs 299.46±6.35 nmol/1) (P>0.05, both); rivaroxaban at 25 nmol/1, however, significantly lowered down ETP (748.42±368.97 vs 1478.65±73.77 nmol-min) and Peak (106.53±55.46 vs 299.46 ±6.35 nmol/1) (P<0.01, both). The IC50s on ETP exhibited significant difference between dabigatran and rivoraxaban (605.5±1.9 vs 24.7±1.1 nmol/l, respectively, ratio:24.5).Conclusion:Dabigatran concentration-dependently delayed the initiation phase of TG, but did not reduce the thrombin burst produced in the propagation phase in lower concentrations (≤100nmol/l). Rivaroxaban not only delayed the initiation phase of TG but also reduced the thrombin burst produced in the propagation phase. Both dabigatran and rivaroxaban concentration-dependently inhibited tissue factor induced-PAgR. The anticoagulation and antiplatelet effects of rivaroxaban were stronger than dabigatran. Co-incubated with rhs-TM can intensified the anticoagulant effect of rivaroxaban but not that of dabigatran.Part Ⅱ:Initiating Anticoagulation with Warfarin in Patients with Atrial Fibrillation:the Tendency of Plasma Endogenous Thrombin Potential. Abstract:Objective:Anticoagulation can improve clinical prognosis in patients with atrial fibrillation at high thromboembolic risk. Indirect evidences have indicated that initiating anticoagulation with warfarin could induce paradoxical hypercoagulability and increased the risk of thromboembolism. The aim of this study is to investigate the endogenous thrombin potential (ETP) in patients with atrial fibrillation initiating anticoagulation with warfarin.Methods:We prospectively recruited 39 patients with atrial fibrillation at high thromboembolic risk without anticoagulation at least 2 weeks. Patients were divided into 2 groups according to the use of low molecular weight heparin (LMWH, 100AxaIU/kg): A, warfarin group,26 cases; and B, co-treated group,13 cases. Group A was subdivided into three subgroups according to the initial dose of warfarin:A1,1.5mg qd,7 cases; A2, 3mg qd.11 cases and A3,6mg qd×2d, and then 3mg qd.8 cases. The basic demographic characteristics, medical histories, blood routine test, d-dimer and fibrinogen were collected. Citrated human whole blood samples were obtained from all patients at baseline and 1,2,3 days after initiating anticoagulation. ETP was tested by calibrated automated thrombogram assay. Other indicators, such as international normalized ratio (INR), activated partial thromboplastin time (APTT), protein C (PC) and protein S (PS) were also detected at corresponding time points. The correlations of ETP and other indicators were evaluated by multivariate linear regression analysis.Results:There were no significant differences in baseline characteristics. INR, APTT, PC, PS and ETP between the groups, except the history of diabetes mellitus (3.8% vs 30.8%, P=0.035). Increasing trends were similar in INR levels (accumulated increase 33.79% vs 34.76%, P=0.947) but dissociated in APTT between the groups, which was gradual increasing in group A, but substantially increased at the 1st day (14.5% vs 3%, P=0.026) and sustained in group B. The accumulated APTT variations within 3 days were approximated between the groups (13.45% vs 15.84%, P=0.628). Both PC and PS activities were gradually decreasing within 3 days, and accumulated variations in PC and PS were no significant differences between the groups (-28.6% vs-23.9%,-18.6% vs-14.5%; P=0.505,0.565, respectively). There was a fluctuation in the decreasing process of ETP in group A, but a linear decrease in group B. No difference of accumulated decrease in ETP were indicated between the groups (-30.5% vs-36.8%, P=0.273). Multivariate linear regression analysis indicated that baseline ETP was independently associated with red blood cell counts (RBC, p=257.6, P=0.001) and INR (β=-1425.8, P=0.001). The variations of ETP at the 1st day and 2nd day were independently associated with the variations of INR (β=-0.946,-0.231; P=0.002,0.004; respectively), and the accumulated variation of ETP within 3 days was independently associated with the variation of PC activity ((3=-0.378, P=0.002). In subgroup analysis, warfarin dose-dependently inhibited ETP. PC and PS. Accumulated decrease of ETP in subgroup Al within 3 days was significantly lower than those in subgroup A2 and A3 (-20.4%,-34.1% and -34.3%, respectively). Decreases of ETP were significantly accelerated at the 3rd day in subgroup A1 and A2 (-8.7% and -21.7%), but at the 1st day in subgroup A3 (-17.5%).Conclusion:Warfarin dose-dependently inhibited ETP, PC and PS. The maximum decrease of ETP was presented at the 3rd day after initiating anticoagulation with low-dose warfarin but at the 1st day with high-dose warfarin. There was a decrease and fluctuation in ETP when initiating anticoagulation with warfarin in patients with atrial fibrillation, which could be smoothed by combining with LMWH. ETP was associated with RBC and INR in patient with atrial fibrillation without anticoagulation. The variations of ETP were associated with those of INR and PC activity when initiating anticoagulation with warfarin.
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