ABSTRACT
Objective To investigate the correlation of interaction between polymorphisms of prothrombin gene G20210A in 3' untranslated region and tissue factor pathway inhibitor (TFPI) gene C399T in 5' untranslated region with thrombin activity in plasma and the pathological stages of esophageal carcinoma.Methods Based on TNM method,we selected 198 patients with stage Ⅰ esophageal carcinoma,198 with stage Ⅱ,198 with stage Ⅲ,and 198 with stage Ⅳ from the First Affiliated Hospital of Xinxiang Medical College from May 2011 to August 2015 for this study;198 patients with esophageal carcinoma of stage 0 served as the control group.The thrombin activity in plasma were determined by chromogenic substrate assay.The genetic polymorphisms of prothrombin gene G20210A in 3' untranslated region and TFPI gene C399T in 5' untranslated region in peripheral blood leukocytes of the above-mentioned patients were analyzed by PCR-RFLP technique.Unconditional logistic regression model and single factor analysis were performed to calculate the adjusted odds ratios (OR) and 95％ confidence intervals (95％ CI) of polymorphisms prothrombin gene G20210A and TFPI gene C399T polymorphisms and to analyze the interaction of nucleotide polymorphisms with thrombin activity in plasma and the pathological stages of esophageal carcinoma.Results The frequencies of G20210A (GA),G20210A (AA),C399T (CT) and C399T (TT) were 24.24％,26.77％,24.24％ and 25.76％ in stage Ⅰ group;34.34％,37.37％,34.85％ and 36.36％ in stage Ⅱ group;39.90％,42.93％,40.41％ and 41.92％ in stage Ⅲ group;45.45％,46.97％,45.35％ and 46.46 in stage Ⅳ group;and 13.64％,14.14％,13.13％ and 13.64％ in stage 0 group,respectively.Statistical tests showed significant difference in the frequencies among each group (all P＜0.01).The risks of invasion and metastasis of esophageal carcinoma significantly increased in the subjects with G20210A,in those with G20210A(AA) genotype,in those with C399T (CT) genotype and in those with C399T (TT) genotype.Combined analysis of the polymorphisms showed that percentage of G20210A (AA)/C399T (TT) in stage Ⅰ group,stage Ⅱ group,stage Ⅲ group,stage Ⅳ group and stage 0 group was 7.07％,14.14％,18.18％,21.71％ and 1.52％,respectively,and statistical tests showed significant difference in the frequency among each group (all P＜0.01).People who carried G20210A(AA)/C399T(TT) had higher risks of invasion and metastasis of esophageal carcinoma,and statistical analysis suggested a positive interaction between G20210A (AA) and C399T (TT) in increasing the risks of invasion and metastasis of esophageal carcinoma (All γ＞ 1).Likewise,there were also positive interactions in the pathogenesis of invasion and metastasis of esophageal carcinoma between G20210A (GA) and C399T (TT),G20210A (GA) and C399T(CT),G20210A (AA) and C399T (CT) (All γ＞1).The thrombin activities in plasma in stage Ⅰ,Ⅱ,Ⅲ and Ⅳ groups were all significantly higher than those in stage 0 group,and there were significant differences among stage Ⅰ,stage Ⅱ,stage Ⅲ and stage Ⅳ in thrombin activities (all P＜0.01).Patients with mutation genotype had significantly higher thrombin activities than those with wild homozygous in the same TNM stage.Conclusion G20210A and C399T gene mutations are the risk factors in the invasion and metastasis of esophageal carcinoma.Significant interactions between G20210A and C399T mutations increase the risk of invasion and metastasis of esophageal carcinoma,which may be closely related to their increased thrombin activities in plasma.
ABSTRACT
To evaluate the effect of different fractions of Taohong Siwu decoction on ADP-induced platelet aggregation and thrombin activity, and to exploit the bioactive constituents, ADP-induced platelet aggregation rate in rabbits was determined by using the method of turbidity method. A bioassay called thrombin time was developed for determining anti-thrombin activities. UHPLC-Q-TOF-MS method was used to qualitatively analyze the chemical constituents of different parts. Alcohol precipitation deposition fraction, alcohol precipitation supernatant fraction and 20% to 30% alcohol elution fraction could significantly inhibit ADP-induced platelet aggregation. Alcohol precipitation supernatant fraction, water insoluble fraction and 40% to 70% alcohol elution fraction could significantly inhibit thrombin activity. The main components of alcohol precipitation deposition fraction, alcohol precipitation supernatant fraction and 20% to 40% alcohol elution fraction were analyzed and identified as aromatic acids, glycosides and phthalides. The bioactive constituents of Taohong Siwu decoction for inhibiting ADP-induced platelet aggregation and thrombin activity include aromatic acids, glycosides and phthalides. This experiment provides scientific basis to further explore the bioactive constituents and mechanism of Taohong Siwu decoction in treating blood stasis syndrome.
ABSTRACT
The effects of harvest and different processing methods on the anti-thrombin activity of Poecilobdella manillensis were respectively studied. The indicators included processing methods (vacuum freeze drying, fresh homogenate, drying under sunlight, freezing, scalding, baking under different temperatures), different parts (entire body, cephalon, pygidium, exudate) and body weights (≤10, 10-20, 20-30, 30-40, ≥40 g). The anti-thrombin activities of P. manillensis with different processing methods were evaluated by direct anti-thrombin titration. The results indicated that the processing methods significantly affected the anti-thrombin activities of P. manillensis. Among the 11 groups, the anti-thrombin activity of P. manillensis processed with vacuum freeze drying (1 303.56 U•g⁻¹) was significantly highest than the other groups (P<0.05), and that processed with baking under 90 ℃ (15.44 U•g⁻¹) was the lowest. The anti-thrombin activity of the cephalon of P. manillensis (226.42 U•g⁻¹) was the highest, and that of the pygidium (102.12 U•g⁻¹) was lowest; the anti-thrombin activities for different body weights were significantly different (P<0.05); and among the five groups, the body weight of ≤10 g (328.86 U•g⁻¹) was the highest (P<0.05), and the body weight of ≥40 g (87.71 U•g⁻¹) was the lowest. In conclusion, harvest and different processing methods had a significant impact on the anti-thrombin activities of P. manillensis. In the study, for the optimal processing method for P. manillensis, the body weight between 20-30 g is recommended, and the vacuum freeze drying is preferred, which is followed by the drying under sunlight.
ABSTRACT
Objective:To compare the anti-thrombin activity and the effects on the coagulation pathway between Whitmania pigra Whitman and Hirudinaria manillensis to provide scientific reference for the anticoagulation mechanism revelation for Hirudo. Methods:Anti-thrombin titration and chromogenic substrate assay-extraction-HPLC were applied to study the anti-thrombin activity of Whitmania pigra Whitman and Hirudinaria manillensis. APTT, PT and TT were determined by a clotting assay to compare the effects on the path-way of blood clotting. Results:The anticoagulation activity order measured by anti-thrombin titration was living Hirudinaria manillensis> dried Hirudinaria manillensis > > dried Whitmania pigra Whitman. The results of chromogenic substrate assay-extraction-HPLC in-dicated that the low dose of aqueous extract promoted the thrombin activity, while the high dose inhibited the thrombin activity. Hirudi-naria manillensis significantly inhibited the activity of thrombin, while Whitmania pigra Whitman showed weak anti-thrombin activity only at the higher dose. All leeches could prolong APTT, PT and TT. However, living Hirudinaria manillensis mainly affected TT, and dried Hirudinaria manillensis mainly affected APTT. Dried Whitmania pigra Whitman dramatically influenced APTT and TT. All the results indicated that the anticoagulant activity of Whitmania pigra Whitmanis was significantly higher than that of Hirudinaria manillen-sis. Conclusion:There are notable differences in the anti-thrombin activity and the effect on the pathway of blood clotting between Whitmania pigra Whitman and Hirudinaria manillensis.