Studies Of Phospholipid-Binding Anticoagulation Protein From Agkistrodon Halys Brevicaudus Venom On Biochemistry, Pharmacology And Molecular Biology

OBJECTIVE Snake venom are complex mixtures containing proteins and polypeptides,which interfere with blood coagulation,fibrinolysis and platelet aggregation.Snake venom are proteins or enzymes,such as L-Amino acid oxidase,phospholipase A ₂,nucleotidase,thrombin like enzymes,fibrinolysin Proteinase C;the others are non-enzymatic proteins, such as RGD-containing single chain polypeptide,carinatin,trigramin, echistatin,applaggin and bitistatin.In this paper we reported the purifcation of Phospholipid-binding anticoagulation protein(PBAP)from Agkistrodon halys brevicaudus venom living in P.R.China and studied its physicochemical proterties;anticoagulation and thrombolytic effects on animal thrombosis;its hemorrheology and general Pharmacology;its acute toxicity and LD₅₀in mice;its cloning and expression and characterization of PBAP in Pichia pastoris.1 METHRODS1.1 The purification and identification of the PBAP from Agkistrodon halys Brevicaudus VenomThe Phospholipid-binding anticoagulation protein was purified from Agkistrodon halys Brevicaudus Venom by Cation ion exchange chromatography on CM Sephadex C-25 and negative ion exchange chromatography on DEAE Sepharose CL-6B,gel filtration on Sephacryl S-200 and Sephadex G-75 chromatography.Its anticoagulant activities in vitro were assayed by activated partial thromboplastin time(APTT);its molecular weight was calculated by SDS-polyacrylamide gel electrophoresis(SDS-PAGE)and its isoelectric point was estimated by the isoelectric focusing electrophoresis.Binding experiments of anticoagulation protein to phospholipids vesicles were performed with Dittmer's thin-layer chromatography.Its N-terminal amino acid sequence was measured by living science Laboratory of peking University using automatic Edman degradation method.1.2 Auticoagulaut effcts of PBAP in vitro on human plasma and in vivo on rabbits1.2.1 To test the activated partial thromboplastin time(APTT),Prothrombin time(PT),Fibrinogen(FIB),Thrombin time(TT)1.2.1.1 In Vitro on human plasmaWhen 0.1ml PBAP(0.2mg/ml)was added into 1,2,4,6,8 ml normal human plasma respectively.Different plasma concentration of 1:10,1:20,1:40,1:60,1:80 were prepared.The APPT,PT,TT,FIB tests were measured by the method of kit illustration on ACL-Advace automatic hemagglutination meter of USA IL company.1.2.1.2 In vivo on rabbits24 rabbits were divided into four groups combining high,middle,low and heparin dosage.The PBAP was given by intravenous injection at 0.2,0.4,0.8 mg/kg respectively and the haparin group was given by 50U/kg.Normal saline was used as the negative control.Before and after administration for 15 minutes,2ml blood from rabbit arteria was drew into plastic tubes containing 3.8%sodium citrate in the 9 to 1 ration.The blood samples were centrifuged at 3000 rpm/min for 10min at 4℃to obtain platelet poor plasma(PPP).The APPT,PT,TT,FIB tests were measured by the method of kit illustration on ACL-Advace automatic hemagglutination meter of USA IL company.1.2.2 Measurement of clotting factor activities 1.2.2.1 In Vitro on human plasmaWhen 0.1ml PBAP(0.2mg/ml)was added into 1,2,4,6,8 ml normal human plasma respectively,different plasma concentration of 1:10,1:20,1:40,1:60,1:80 were prepared.The Fâ…¡:C,Fâ…¤:C,Fâ…¦:C,Fâ…§:C,Fâ…¨:C,Fâ…©:C,Fâ…ª:C,Fâ…«:C activity tests were determined by a standard assay established in Triplett using congenitally deficient plasma according to the manufacture's instruction on ACL-Advace automatic hemagglutination meter.1.2.2.2 In vivo on rabbits24 rabbits were divided into four groups combining high,middle,low and heparin dosage.The PBAP was given by intravenous injection at 0.2,0.4,0.8 mg/kg respectively and the haparin group was given by 50U/kg.Normal saline was used as the negative control.Before and after administration PBAP for 15 minutes,2ml blood from rabbit arteria was drew into plastic tubes containing 3.8%sodium citrate in a 9 to 1 ration.the blood samples were centrifuged at 3000 rpm/min for 10min at 4℃to obtain platelet poor plasma(PPP).The Fâ…¡:C,Fâ…¤:C,Fâ…¦:C,Fâ…§:C,Fâ…¨:C,Fâ…©:C,Fâ…ª:C,Fâ…«:C activity tests were determined by a standard assay established in Triplett using congenitally deficient plasma according to the manufacture's instruction on ACL-Advace automatic hemagglutination meter.1.2.3 Tissue Thromboplastin Inhibition(TTI)test,Dilute Russell's Viper Venom Time(dRVVT)TestThe TTI test was peformed according to the method of Schleider and TPL was used at 1:1,1:100,1:1000 dilution in quadruplicate for each dilution.The dRVVT test was done in quadruplicate using commercial kits acoording to the manufacturer's instruction.1.3 The thrombolytic effect of PBAP purified from Agkistrodon halys brevicaudus venom on animalthrombosisBy means of Nowork's pulmonary embolism model in rabbits and Reyers's venous thrombosis model in rats,the PBAP was given by intravenous injection at 0.2,0.4,0.8mg /kg three different doses or one dosage respectively.Normal saline was used as the negative control and thrombolytic effects were observed and Chandler's mixed thrombosis model in vitro,10,30,50μl PBAP(0.62mg/ml)was added into plastic tubes containing 1ml rabbit blood.The weight and length of the thrombi were observed.1.4 The effect of PBAP from Agkistrodon HalTs Brevicaudus Venom on hemorrheologyThe rabbits were given by intravenous injection at 0.2,0.4mg/kg PBAP respectively.Normal saline was used as the negative control,heparin(100U/kg) was used as the positive control.Before and after 1h,the whole blood viscosity,the blood plasma viscosity and the hematocrit of red blood cell were measured by LBY-N6A self erasure rolary viscometer.1.5 The Effect of PBAP from Agkistrodon Halys Brevicaudus Venom on the General Pharmacology1.5.1 After mice were given by tail intravenous injection 0.4mg/kg PBAP. their general behavior,coordination,synergism with sodium pentobarbital at subthreshold were observed.1.5.2 After rabbits were given by iv 0.4mg/kg PBAP,rabbit's heart rate, electrocardiogram(ECG),average arterial pressure,breath frequency were observed by BL-420 biology function experiment system.1.6 The acute toxicity and LD₅₀of PBAP from Agkistrodon Halys Brevicaudus VenomBy means of Bliss method,the LD₅₀and its 95%confident limit on mice were calculated by ip and iv PBAP.1.7 Molecular clone and sequence analysis of cDNA encoding of Phospholipid-binding anticoagulation protein from Agkistrodon halys Brevicaudus Venom The total mRNA was extracted from the venom gland of Agkistrodon halys Brevicaudus.The cDNAs encoding of PBAP were amplified by RT-PCR using different primers.The 400bp PCR product was cloned into the pMD 19-T Vector.Plasmid DNA obtained from positive clones was identified by means of digestion with EcoRâ… and PCR reaction.The cDNA sequences were determined with positive clones or purified PCR products directly.2 RESULTS2.1 The purification and identification of Phospholipid-binding anticoagulation protein from Agkistrodon halys Brevicaudus VenomBy means of CM-Sephadex C-25,DEAE-Sephadex A-50,Sephadex G-200 and Sephadex G-75 chromatographies.A kind of Phospholipid-binding anticoagulation protein from Agkistrodon halys Brevicaudus Venom(PBAP) was purified.On SDS-PAGE,the purified PBAP had a molecular weight of 24.0 X 10³ under non-reducing condition and 14.6 X 10³ under reducing condition.We found that its contents of Acidic animo acid(aspartic acid,glutamic acid)were more than alkalinous animo acid.The isoelectric point was pH 5.2 by the isoelectric focusing electrophoresis.Its N-terminal amino acid sequencing revealed a mixture of 2 sequences:D-C-P-S-(D/G)-W-S-S-Y-E-G-H-C-Y-(Q/K).Having arginine ester-hydrolyzing enzyme and binding Phospholipid.2.2 The auticoagulaut effcts of PBAP heve showed in vitro on human plasma and in vivo on rabbits2.2.1 PBAP can markedly prolong APTTWhen three plasma concentrations of 1:40,1:60,1:80 were tested,the APTT was 57.54±2.66,74.77±7.18,107.92±11.4(S)respectively. Compared with the control group(37.43±2.03 S),APTT was prolonged markedly(P＜0.01).The prolongation of APTT was depended on the concentration of PBAP.The higher concentration of PBAP,the stronger anticoagulant effects and their effects were concentration-dependent but time-independent.The anticoagulaut effcts of PBAP on rabbits were indicated in vivo:after administration 0.8,0.4,0.2mg/kg PBAP for 15min,The APTT was 116.94±38.19,87.27±7.13,76.07±11.66(S),respectively.Compared with the before administration(53.82±1.96,54.77±5.75,57.95±12.24 Second),APTT was markedly prolonged and statistic significant difference(P＜0.01 or 0.05).2.2.2 TTI and dRVVT testsWhen APTT reagent was used at 1:100 and 1:1000 dilution at 4.4μg/ml PBAP,In TTI test,The APPT was 54.7 second(control 48.7 second)and 176.8 second(control 137.7 second).It was clearly shown that PBAP had obvious effects on TTI in higher dilution of APTT reagent,PBAP could accentuate the inhibitor effect by decreasing phospholipid concentration. In dRVVT test,dRVVT was 47.7 second and 56.5 second(control 41.5 second)when PBAP dose was 2.2μg/ml PNP and 4.4μg/ml PNP,respectivively.Suggesting dRVVT was prolonged in the presence of PBAP compared with the control(41.5 second)and it was dose-dependent.2.2.3 The Fâ…§:C,Fâ…¨:C,Fâ…©â… :C,Fâ…©â…¡:C activity was inhibited by PBAPIn vitro the clotting factors tests indicated that PBAP could markedly inhibit Fâ…§:C,Fâ…¨:C,Fâ…©â… :C,Fâ…©â…¡:C activities when PBAP was assayed at concentration of 1:10,1:20,1:40 ditution plasma(P＜0.01)and only Fâ…¨:C activity was inhibited at concentration of 1:60,1:80 ditution plasma and in vivo the clotting factor tests on rabbits indicated that PBAP could inhibit Fâ…§:C,Fâ…¨:C,Fâ…©â… :C,Fâ…©â…¡:C activities after administration 0.8,0.4mg/kg PBAP for 15min and it was concentration-dependently.(p＜0.01).2.3 The thrombolytic effect of PBAP from Agkistrodon halys brevicaudus venom on experimental thrombosis of animal2.3.1 On mixed thrombosis by chandle's method When 10,30,50μl PBAP(0.62mg/ml)was added into plastic tubes containing 1ml rabbit blood,the dry weights of the thrombi were reduced to 0±0,15.4±2.2,18.5±1.9mg from 23.7±2.1(control)(p＜0.01)and the moist weight were reduced to 0±0,72.9±12.3,96.4±12.1 mg from 132.7±10.5(control)(p＜0.01).10 rabbits were injected i.v with 0.8,0.4,0.2 mg/kg PBAP for 5,15,30,45min,the dry weight and moist weight of the thrombi after administration of PBAP were lighter than those of untreated(P＜0.05-0.01)and reduced markedly afer administration 5 min.2.3.2 On venous thrombosis by Reyer's methodThe incidence of thrombus formation and thrombus moist mean weight 2 hours after ligation of inferior vena cava were 100%and 8.03±2.48 mg respectively in cotrol group of 10 rats,while the PBAP group of 10 rats were 0%and 0±0 mg(P＜0.01).2.3.3 On pulmonary embolism by Nowork's method20 rabbits were introduced with external emboli through cervical vein into general circulation.10 rabbits were given 0.4mg/kg PBAP and 10 control rabbits were given normal saline.24 hours later,the weight of the former emboli decreased from 32.26±2.23mg to 4.46±0.55mg and it has statistical significance(p＜0.01),compared with normal saline.2.4 The viscosity of whole blood and hematocrit of red blood cells in rabbits were reduced by The PBAPAfter intravenous injection of PBAP(0.4mg/kg)for 1h,the whole blood viscosity of 10,50,150(1/S)on rabbits was decreased 0.91±0.31,0.51±0.11, 0.39±0.08 respectively(P＜0.05)and the hematocrit of red blood cell of 10,50, 150(1/S)on rabbits was decreased 4.00±1.41%(P＜0.01),compared with control.2.5 The Effect of PBAP from Agkistrodon Halys Brevicaudus Venom on the General Pharmacology All animal groups of mice or rabbits had shown that PBAP had no influence on their general behavior,coordinate activity and synergism with sodium pentobarbital at subthreshold dose and the nervous,cardiovascular and respiratory systems after administrated at different doses of PBAP were not changed,compared with control.2.6 Acute toxicity tests of PBAP from agkistrodon halys brevieaudus venomLD₅₀and 95%confident limit on mouse were 26.869mg/kg and 23.348～30.65mg/kg by iv PBAP,LD₅₀and 95%confident limit were 39.897mg/kg and 35.948～44.128mg/kg by ip PBAP.2.7 Molecular cloning and sequence analysis of cDNA encoding of Phospholipid-binding anticoagulation protein from Agkistrodon halys Brevicaudus VenomThe cDNA sequence with 599 bp encodes an uncompleted open reading frame(ORF)of 146 amino acids,which included a signal peptide of 23 amino acids,mature protein of 123 amino acids.Compared with the amino acids sequence for halyxin B-chain precursor[Gloydius halys],Coagulation factorâ…¨/factorâ…©-binding proten B chain precursor,ACF 1/2 B-chain [Deinagkistrodon acutus],fibrinogen clotting inhibitor B chain[Gloydius halys brevicaudus],factorâ…¨/â…©binding protein beta chain-2[Trimeresurus stejnegeri],anticoagulant protein-B[Deinagkistrodon acutus],factorâ…¨/â…©binding protein beta chain-1[Trimeresurus stejnegeri],coagulation factorâ…¨-binding protein chain B[Gloydius halys],Their homology were 95%,89%,89%,90%,88%,88%,88%,99%.3 CONCLUSIONThe Phospholipid-binding anticoagulation protein from the Agkistrodon halys Brevicaudus Venom(PBAP)was a dimer and its molecular weight was 24.0 X 10³ under non-reducing condition and 14.6 X 10³ under reducing condition.The isoelectric point was pH 5.2 by the isoelectric focusing electrophoresis.PBAP also had arginine ester-hydrolyzing enzyme activity and binding Phospholipid.In the presence of PBAP the APTT and Dilute Russell's Viper Venom Time(dRVVT)of human plasma were markedly prolonged(P＜0.01) and their effects were concentration-dependent but time-independent.The inhibitory effect of PBAP on the plasma clotting time was enhanced by decrasing phospholipis cocentration in Tissue Thromboplastin Inhibition(TTI) test.It had anticoagulation activity by means of prolonging on APTT and inhibiting Fâ…§:C,Fâ…¨:C,Fâ…©â… :C,Fâ…©â…¡:C activity and obvious thrombolytic effect on experimental thrombosis of animals.The PBAP was able to reduce the viscosity of whole blood and hematocrit of red blood cells in rabbits,but it had no effects on nervous,cardiovascular and respiratory systems.The toxicity of PBAP was lower and non-haemorrhage and non-haemolysis.It was a effective modern antithrombotic compound with potential clinical values.Compared with the amino acids sequence for other C-lectin of snake venom.Their homology were 88%—99%.