| In the blood,there is a dynamic balance between coagulation system and fibrinolytic system to maintain blood flow normally.The coagulation system include coagulation and anticoagulation.So as the consists of fibrinolytic system are fibrinolysis and anti fibrinolytic.If this dynamic balance were destroyed,it could lead to thromboembolic disease,or cause hemorrhagic disease.The clinical treatment of thromboembolic diseases drugs currently include anticoagulant,antiplatelet drugs and fibrinolytic drugs at home and abroad.Peptide drugs(less than 100 amino acids)are developing quickly because it has an effective and quick action with low side effects,like hirudin,bivalirudin and oral peptide drugs(ximelagatran),which will lead the development direction of anticoagulant thrombolytic drugs in the future.The new anticoagulant and thrombolytic drugs can not only play a role in thrombolytic therapy,but also promote the fibrinolytic pathway.This research focus on the effect of Hirulog-s,a novel stearic acid-modified hirudin peptidomimetic,about fibrinolytic kinetics in vitro and thrombolytic activities in vivo.At the same time,the fibrin activity of Hirulog-s was studied that could reflect the risk of bleeding.Finally,paper evaluated the pharmacological effects of Hirulog-s on fibrinolytic system and put some suggestions.The first chapter summarizes the action mechanism and clinical characteristics of thrombin inhibitor in thromboembolic disease.The targeting of thrombin inhibitor is thrombin,which is one of the effective methods of clinical treatment of thrombosis.According to the mechanism of action,thrombin inhibitors could be divided into indirect thrombin inhibitors and direct thrombin inhibitors.Though indirect thrombin inhibitors,such as heparin and Hua Falin,have some application value in the treatment of thrombosis,they have some limitations.The other drugsthat direct thrombin inhibitors have better clinical effects than heparin drugs also have some disadvantages.So researcher began to study the new anticoagulant thrombolytic drugs.In recent years,many direct thrombin inhibitors that used advanced biological engineering technology have been successfully developed in clinical trails,and have more effects than traditional anticoagulant drugs.There are many researches about mechanism and clinical effect of the new thrombin inhibitors need to be studied deeply.The second chapter will comprehensively introduce the influence of Hirulog-s in mutual activation reaction system constituted by single chain urokinase-type plasminogen activator(pro-uPA)and plasminogen(plg).The enzymatic reaction kinetic constant(Km,kcat)of plasmin(plm)acts on pro-uPA in mutual activation reaction system influenced by pro-uPA and plg was measured.And the impact of Hirulog-s on the catalytic reaction activity and affinity by comparing kinetic constants of the enzymatic reaction in different concentrations of Hirulog-s was investigated as well.The role of fibrinolytic activity was detected by enzyme substrate reaction.And the method was used to determine the kinetic constant of the reaction of plasminogen activation which was catalyzed by single chain urokinase-type plasminogen activator.The kcat value of Hirulog-s(30μmol·L-1)was 3.1 times more than the control group in the in the pro-uPA reaction system influenced by plm.The Km value of Hirulog-s(30μmol·L-1)was 1.6 times lower than that of control group.The results indicated that Hirulog-s has increased the maximum rate of catalysis and affinity significantly.Compared to the control group,the kcat/Km value of Hirulog-s(30μmol·L-1)was 4.75 times increased.That means Hirulog-s greatly improved the catalytic efficiency of the plm and has stronger reactivity than Hirulog-1.The Hirulog-s could influence fibrinolysis process by promoting mutual activation reaction between pro-uPA and plg.The novel synthetic hirudin significantly enhanced the kinetic constant of fibrinolytic activation.The third chapter emphasizes the thrombolytic effect of Hirulog-s.A criteria that is based on fibrinolysis experiment in vitro to evaluate the thrombolytic effect of Hirulog-s in vivo.Firstly,the experiment establish a FITC-pulmonary thrombosis model in rats.Moreover,rats are random to divided groups as follow: saline group as control group;pro-uPA drugs as positive control group;high,medium and low dose of Hirulog-s groups were treated;high,medium and low dose of Hirulog-1 groups as control groups.After administration of 12 h,the rats were dissected to take blood from heart and take lung.The fluorescence intensity of the plasma was detected and the lung sections were observed,which could evaluate the thrombolytic effect of high,medium and low doses of Hirulog-s.The results showed that lung tissue slices injected with 1.6 mg/kg·bw of Hirulog-s indicated alveolar structure integrity,alveolar wall uniformity,remnants of pulmonary embolism after dissolution of the traces in acute pulmonary thromboembolism model.It is similar to pro-uPA positive control.The effect of Hirulog-1 was not obvious through the lung sections,and the effect was not as good as Hirulog-s.Concurrently,the fluorescence intensity of 1.6 mg/kg·bw group in plasma of rats was significantly higher than the control group.Based on observation,Hirulog-s shared the same fibrinolysis effect as that of pro-uPA.Chapter four focuses on the influence of Hirulog-s on the activity of fibrous proteins of wistars.The same groups as follow: saline group as control group;pro-uPA drugs as positive control group;high,medium and low dose of Hirulog-s groups were treated.After injecting drugs into wistars via caudal veins,the calcium addition method was adopted to test the dissolve time of the euglobulin in blood.The ELISA was used to investigate the content of fibrinogens and their split products.An analysis on dates revealed that the dissolve time was indeed shorten by 32 to 40 seconds in 120 minutes after injection of Hirulog-s.The marked difference(p<0.05)was observed between the Hirulog-s group and the control group,which demonstrated that Hirulog-s exactly increased the activity of fibrinolysis in plasma.In addition,the content of fibrinogens was marked difference of 0.4 mg/kg·bw group,0.8 mg/kg·bw group,1.6 mg/kg·bw group comparing with the control group.But their split products were not showed marked difference.It is suggested that the decrease of fibrinogen in the blood is not because the excessive fibrinolysis,while the decrease of fibrinogen may lead to partial hemorrhage. |
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