Antithrombotic Effect Of A Plasminogen Activator From Gloydius Brevicaudus Venom

Snake venoms are complex mixtures containing many different biologically active proteins and peptides. A number of these proteins interact with components of the human hemostatic system.In 1993,A novel plasminogen activator form Trimeresurus stejnegeri venom(TSV—PA) has been identified and purified by Yun Zhang for the first time.It offers the new subject for developing new medicine from venom.This study was designed to isolate and purify novel plasminogen activator from Gloydius brevicaudus venom(GBV)and analyse its characterization and biological activities.1. Purification of the plasminogen activator from Gloydius brevicaudus Venom. Affinity chromatography of Gloydius brevicaudus Venom in Benzamidine Sepharose 6B resulted in the separation of two protein peaks. GBV-PA was purified by Lichrospher C18 4.6/250 reverse chromatography. The fractionⅡcan specifically activate plasminogen through an enzymatic reaction estimated by the fibrin plate method. It was homogeneous as a single band showed on SDS-polyacrylmide gel electrophoresis (SDS-PAGE,Tris system).2. Thrombolytic effect of GBV-PA in rats common carotid artery of thrombosis. Thrombogenesis was induced by FeC13, thrombogenesis and thrombolysis were judged by temperature of lingual root of rats. GBV-PA 150,300μg·kg-1 iv could upgrade the temperature of lingual root of rats, the effect continued more than 420 minutes; UK 2000IU·kg-1 iv also upgraded the temperature of lingual root of rats , but the effect attenuated after 180 minutes.Comparing with the thrombus weight, GBV-PA 150,300μg·kg-1 were 25.3±0.06mg and 17.6±0.73mg respectively,UK was 22.9±0.73mg,with obvious difference compared with NS (p<0.01). GBV-PA has the thrombolytic effect to thrombus of rats common carotid artery, and persists longer time compared with UK.3. Protective effect of GBV-PA in rats common carotid artery of thrombosis . Thrombogenesis was induced by FeC13 .The drug was given 60 minutes before spreading filter paper of FeC13 . GBV-PA 150,300μg·kg-1 iv could inhibit the degrad of temperature of rats lingual root. Comparing with the thrombus weight, GBV-PA 150,300μg·kg-1 were 21.5±0.13mg and 15.2±0.69mg respectively,UK was 20.6±0.33mg,with obvious difference compared with NS (p<0.05). GBV-PA has the inhibition to thrombosis of rats common carotid artery.4. Effect of GBV-PA on thrombolysis of rats in man-made artery-vein circulation.The effect of GBV-PA was estimated by weight of thrombus.GBV-PA 150,300μg·kg-1 iv could significantly reduce the weight of thrombus.Comparing with the thrombus weight, GBV-PA 150,300μg·kg-1 were 23.3±1.57mg and 14.7±0.66mg respectively,asprin was 14.9±0.90mg,with obvious difference compared with NS (p<0.01). GBV-PA has the inhibition to thrombosis of rats common carotid artery in man-made artery-vein circulation.5. Thrombolytic effect of GBV-PA on thrombus of inferior caval vein induced by power of rabbit brain.The thrombus of inferior caval vein was induced by power of rabbit brain. GBV-PA 150,300μg·kg-1 iv could significantly reduce the weight of thrombus.Comparing with the thrombus weight, GBV-PA 150,300μg·kg-1 were 20.3±2.15mg and 12.5±1.44mg respectively,asprin was 16.2±3.15mg,with obvious difference compared with NS (p<0.01). GBV-PA has the inhibition to thrombosis of rats inferior caval vein induced by power of rabbit brain.6. Protective effect of GBV-PA on rats with pulmonary thrombus.We observed the effect on the respiratory distress due to pulmonary thrombosis induced by ADP-Na2 after GBV-PA 150 and 300μg·kg-1 iv in rats.Comparing with time of respiratory distress, GBV-PA 150,300μg·kg-1 were 189.3±12.4s and 131.2±12.0s respectively,asprin was 159.9±14.2s,with obvious difference compared with NS (p<0.01). GBV-PA markedly relieved the respiratory distress.7. Thrombolytic effect of GBV-PA on dogs with cerebral thrombus.The model of internal cerebral embolism was established with interventional technique in 19 beagle adult dogs,which were randomly divided into 5 groups including control group, group of 104IU·kg-1 dose of rt-PA, group of 18.75μg·kg-1 dose,37.5μg·kg-1 dose,75μg·kg-1 dose of GBV-PA. Angiography was performed half,1,2 and 3 hours after thrombolysis to observe recanalization. The rate of recanalization 1 hour after administration of rt-PA,low dose,middle dose,high dose of GBV-PA were 100%,0%,25%and 50%;2 hours were 100%,0%,50% and 75%;3 hours were 100%,50%,75% and 100%,showed significanlly statistic difference compared with control group(p<0.05). There were time-response and dose-response relationships when the cerebral infarction beagle dogs were treated with GBV-PA. ConclusionsGBV-PA 150,300μg·kg-1 iv have the thrombolytic effect to thrombus of rats common carotid artery induced by FeC13, and persists longer time compared with UK.;GBV-PA 150,300μg·kg-1 iv have the inhibition to thrombosis of rats common carotid artery induced by FeC13; GBV-PA 150,300μg·kg-1 iv have the inhibition to thrombosis of rats common carotid artery in man-made artery-vein circulation; GBV-PA 150,300μg·kg-1 iv have the inhibition to thrombosis of rats inferior caval vein induced by power of rabbit brain; GBV-PA 150,300μg·kg-1 iv relieve the respiratory distress; GBV-PA 150,300μg·kg-1 iv have thrombolytic effect to beagle dogs with cerebral thrombus, there are time-response and dose-response relationships when the cerebral infarction beagle dogs are treated with GBV-PA.