Effect Of Plasminogen Activator From Gloydius Brevicaudus Venomon On Animal Blood Coagulation, Circulatory, Respiratory And Central Nervous System Function

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. Our laboratory has purified novel plasminogen activator from Gloydius brevicaudus venom(GBV)and analyse its characterization and biological activities by animal experiment, and the results shown that the GBV-PA can treated and protected the thrombus in animal. In order to analyse its metabolic process in vivo, this study was designed to isolate and purify GBV-PA and study its pharmacokinetic, as a basic data for its further clinical study.1. Purification and characterization of the plasminogen activator from Gloydius brevicaudus Venom.1.1 Purification of GBV-PAAffinity chromatography of Gloydius brevicaudus Venom in Benzamide SepharoseTM 4 Fast Flow(high sub)resulted in the separation of two protein peaks. The fractionⅡwas collected and purify though gel filtration of Superdex G-75, which yielded three protein peaks. The descending branch of fractionⅡcan specifically activate plasminogen through an enzymatic reaction estimated by the fibrin plate method. GBV-PA was purified from the fractionⅡby Lichrospher C18 4.6/250 reverse chromatography.1.2 The characterization of GBV-PAHomogenicity of GBV-PA was tested by SDS-PAGE and meanwhile molecular weight of it was estimating also. The image of SDS-PAGE showed one band both in native sample of GBV-PA and denaturing sample of GBV-PA with molecular weight of 40kDa. 1.3 The Acute toxicity of GBV-PA in miceResearchers Determine the Acute toxicity of GBV-PA in mice According with the technical guidelines of Acute toxicity test of chemical substances.The acute toxicity does not occur in mice with an initial dose (360μg.kg-1) referencing to the relevant literature.increasing doses of a multiple of two times, the acute toxicity in mice don"t appears in 128 times of initial dose(360μg.kg-1). Carrying out the maximum tolerated dose (Maximal tolerance dose, MTD) experiments, mice were still no other abnormal reaction. result Show that the mice LD50 can not be obtained by administer intravenously GBV-PA, single dose have no significant toxicity.2 The effect of GBV-PA on coagulation in rabbitTaking the healthy rabbits 30, male and female in half, divided into 5 groups : GBV-PA 75μg.kg-1, 150μg.kg-1, 300μg.kg-1, physiological saline group and the UK group. PT, APTT, TT ,and clotting time (CT) of rabbit plasma was tested before and after treatment with GBV-PA,NS or UK for 60 min. Compare with the Premedication and NS group, the CT in rabbits extend from the original 108s to 120s ~ 158s,the APTT in rabbits extend from the original 40s to 68s ~ 153s,the PT extend from the original 16s to the 48s ~ 93s,and the TT extend from the original 20s to around 98s ~ 150s.3 The effect of GBV-PA on circulatory, respiratory and central nervous system function3.1 The effect of GBV-PA on the nervous system of miceObservation the effects of GBV-PA in the autonomous behavioral activity, spontaneous activity, the coordination of movement and the valve under the sodium pentobarbital hypnosis experiment effects in mice have no obvious effect.3.2 The effect of GBV-PA on blood pressure and heart rateThe rat systolic blood pressure (SAP), diastolic blood pressure (DAP), heart rate (HR) was studied before and after administration of GBV-PA.compare with the NS control group,GBV-PA had no effect on SAP, DAP, MAP and HR of rat.3.3 The effect of GBV-PA on the respiratory system in anesthetized rats.Cable tension were Connection with animal skin Xiphoid Department using Suture,the signal connected to the multi-channel bio-signal recording device measuring the respiratory rate and respiratory rate of anesthetized rats before and after administration of GBV-PA,while determination of major mouse ECG usingⅡECG Electrode. Results Compare with the control group, GBV-PA had no effect on the rat respiratory amplitude, frequency and ECG of rat.Conclusions LD50 of GBV-PA Couldn't be obtained by administer intravenously in mice.It had no significant toxicity in mice.GBV-PA iv 75μg.kg-1, 150μg.kg-1, 300μg.kg-1 significant impact on rabbit plasma PT, APTT, TT, CT..GBV-PA iv 90,360,720μg.kg-1don't have obviously effect on independent behavioral activity, coordinated movement or other neurological and psychiatric system activity. GBV-PA iv75,150,300μg.kg-1 don't have significant effect on rat respiratory amplitude,respiratory frequency and ECG.