Purification, Anti-thrombus Effect And Expression Of A Plasminogenactivator From Gloydius Brevicaudus Venom

ObjectiveThromboembolic disease,such as Myocardial infarction,stroke and deep vein thrombosis,does great harm to people's health. The most common thrombolytic agents have been streptokinase,urokinase and reteplase. But most of them have short half-life.In the quest for new thrombolytic agents, scientists turned to extracting natural thrombolytic agents from animals and insects and from snake venoms. Snake venoms were complex mixtures containing many different biologically active proteins and peptides. A number of these proteins act on components of the haemostatic system in humans.This study was designed to isolate and purify novel plasminogen activator from Gloydius brevicaudus venom(GBV-PA)and analyse its characterization and biological activities.MethodA novel plasminogen activator from Gloydius brevicaudus venom was identified and purified to homogeneity by Affinity chromatography and RP-HPLC. The biological activity of GBV-PA was assayed by The fibrin plate method. molecular weight and isoelectric point were assayed by SDS-PAGE and disc polyacrylamide gel eletrophoresis. The biological activities were assayed by the chromogenic substrate method and Fibrin binding experiments. The study on thrombolytic effect of GBV-PA by using three models which were thrombus of common carotid artery induced by FeC13 in rat,and thrombus of inferior caval vein induced by powder of rabbit brain and cerebral thrombus in dog. The platelet aggregation induced by ADP and thrombin were assayed by the Born's method. The effect on t platelet aggregation was observed by the model of pulmonary thrombus in rats and thrombus of man-made artery-vein circulation in rat. Isolated the complementary DNA encoding PA precursor from the Gloydius brevicaudus By RT-PCR. The cDNA of GBV-PA were subcloned into PET-42a expression vector and then transfected into Escherichia coli. The inclusion of the transfected cells turned out biological activity of GBV-PA as assayed by The fibrin plate method.ResultsA novel plasminogen activator from Gloydius brevicaudus venom has been identified and purified to homogeneity by Affinity chromatography and RP-HPLC. SDS-PAGE shows that GBV-PA was homogeneous as a single band in non-reducing conditions and reducing conditions.It is a single chain protein with an apparent molecular weight of 32600 and an isoelectric point of pH 5.2. GBV-PA specifically activates plasminogen through an enzymatic reaction that leads to plasmin generation and displays an indirect fibrinolysis activity through using the chromogenic substrate method. The activity of GBV-PA was 2.87 t-PA IU·mg-1 according the chromogenic substrate S- 2390. The activity of GBV-PA was not affected by a metal chelator (EDTA).In the contrast, PMSF, the irreversible serine protease inhibitor,can completely inhibite the fibrinolysis activity of GBV-PA. These observations show that GBV-PA is not a metallo-protease and indicated that it is a serine protease. Fibrin binding experiments demonstrate that GBV-PA has not fibrin binding capacity.The study on thrombolytic effect of GBV-PA by using three models .The results indicated that GBV-PA have the thrombolytic effect which are time-response and dose-response relationships.And it also can inhibit the formation of thrombus. According to the Born's method,the effects of GBV-PA on platelet aggregation induced by ADP and thrombin were assayed. The results showed that GBV-PA dose-dependently inhibited ADP- induced and thrombin-induced platelet aggregation.We also certified that GBV-PA can inhibit the accumulation of platelet by the model of pulmonary thrombus in rats and thrombus of man-made artery-vein circulation in rat.By RT-PCR, we successfully isolated the complementary DNA encoding PA precursor from the Gloydius brevicaudus. Sequence analysis revealed the obtained DNA consisted of 777bp .The nucleotide sequence which presents96% and 91% homology to that of Agkistrodon halys and Trimeresurus Stejnegeri respectively. The cDNA inserts of GBV-PA were subcloned into PET-42a expression vector and then transfected into Escherichia coli. SDS - PAGE showed that the fusion protein was expressed as inclusion body formation in E. coli BL21(DE3). The rGBV-PA fusion protein was obtained by His-bind Purification system,which has the antigenicity of Agkistrodon halys pallas venom.ConclusionsIn summary, A novel plasminogen activator from Gloydius brevicaudus venom has been identified and purified to homogeneity by Affinity chromatography and HPLC.It is a single chain serine proteases with an apparent molecular weight of 32600 and an isoelectric point of pH 5.2,but it has not fibrin binding capacity .It can specifically activates plasminogen to plasmin generation and displays an indirect fibrinolysis activity. It has the thrombolylic effect of both venous and arterial thrombosis . It can inhibite platelet aggregation and the thrombus formulation. a novel plasminogen activator of Gloydius brevicaudus had been successfully over expressed in Escherichia col.The obtained protein can be used as an instrument under investigation of pharmacokinetics and pharmacodynamics of GBV-PA.